fenretinide and Carcinoma--Squamous-Cell

Chemoprevention is one of the cancer prevention methods, applied for the oral squamous cell carcinoma and its main precursor lesions--leukoplakia and erythroplakia. Presently, the most extensive clinically studied group used in such cases are retinoids: vitamin A (retinol), 13-cis-retinic acid (isotretinoin), N-(4-hydroxyphenyl)retinamide (fenretinide) and precursor of vitamin A--beta-carotene. However, despite good short-time effectiveness, retinoids do not prevent recurrences of the lesions and insignificantly increase cancer-free survival. Moreover, they are also characterized by relatively high toxicity. Vitamin E, Bowman-Birkprotease inhibitor, Spirulina fusiformis and green tee extracts as well as traditional Chinese herbs known as ZengShengPing were also found as effective agents. Lack of activity was reported for cyclooxygenase inhibitors--ketorolac and celecoxib. More promising data was collected from animal experimental studies with chemically induced oral squamous cell carcinoma. Chemopreventive activity was revealed for various agents including plant-derived compounds like resveratrol, green and black tee polyphenols, as well as protocatechuic, ellagic and caffeic acids.

Topics: Animals; Antineoplastic Agents; beta Carotene; Carcinoma, Squamous Cell; Chemoprevention; Drugs, Chinese Herbal; Fenretinide; Humans; Isotretinoin; Mouth Neoplasms; Neoplasm Recurrence, Local; Phytotherapy; Precancerous Conditions; Vitamin A

Basement membrane invasion defines malignant transformation of surface premalignancy. Treatment of oral squamous cell carcinoma (OSCC) cells with the synthetic vitamin A derivative, fenretinide (4HPR), induces numerous cancer-preventive effects including suppression of basement membrane invasion, elimination of anchorage-independent growth, disruption of actin cytoskeletal components and inhibition of the invasion-enabling focal adhesive kinase. The purpose of this study was to elucidate 4HPR's effects on additional invasion-relevant mechanisms including matrix metalloproteinase (MMP) activation and function, cell-extracellular matrix (ECM) attachments and interaction with a kinase that is essential for the epithelial-myoepithelial transformation i.e. c-Jun NH2-terminal kinase (JNK). Our data revealed that 4HPR binds with high affinity to the ATP-binding site of all three JNK isoforms with concurrent suppression of kinase function. Additional studies showed 4HPR treatment inhibited both OSCC cell-ECM adhesion and MMP activation and function. JNK downregulation and induced expression studies confirmed that the JNK3 isoform conveyed that largest impact on OSCC migration and invasion. Biodegradable polymeric implants formulated to preserve 4HPR's function and bioavailability were employed to assess 4HPR's chemopreventive impact on an OSCC tumor induction model. These studies revealed 4HPR local delivery significantly inhibited OSCC tumor size, mitotic indices and expression of the endothelial marker, erythroblast transformation-specific-related gene with concurrent increases in tumor apoptosis (cleaved caspase-3). Collectively, these data show that 4HPR suppresses invasion at multiple sites including 'outside-in' signaling, cell-ECM interactions and suppression of MMPs. These functions are also essential for physiologic function. Regulation is therefore essential and reinforces the pharmacologic advantage of local delivery chemopreventive formulations. .

Topics: Actins; Adenosine Triphosphate; Carcinoma, Squamous Cell; Caspase 3; Cell Line, Tumor; Extracellular Matrix; Fenretinide; Head and Neck Neoplasms; Humans; Matrix Metalloproteinases; Mouth Neoplasms; Neoplasm Invasiveness; Squamous Cell Carcinoma of Head and Neck; Vitamin A

Local, long-acting release fenretinide (4HPR) millicylindrical implants were prepared and evaluated for their release kinetics in vivo and their ability to suppress oral cancer tumor explant growth. Poly(lactic-co-glycolic acid)(PLGA) implants were prepared as a function of drug loading and the presence of various excipients (pore-formers, solubilizers, crystallization inhibitors) to enhance release of the insoluble 4HPR. Release kinetics and bioerosion of PLGA were monitored both in vitro in a PBS/Tween 80 buffer and in vivo by recovery of the drug remaining at the injection site. 4HPR was released from PLGA implants much slower in vivo than in the drug solubilizing media in vitro, with a 3-week lag phase and continuous release of >2 months, but showed some release enhancement by addition of solubilizers. Water-soluble PVA/sucrose implants for release of 4HPR served to determine if drug dissolution provided suitable controlled release without the PLGA, and this formulation showed continuous drug release over 6 weeks in vivo. Placement of PLGA-4HPR implants adjacent to oral cancer tumor murine xenografts showed inhibition of tumor growth relative to sham implants, indicating the potential for the local 4HPR delivery approach to be useful for oral cancer chemoprevention.

Topics: Animals; Anticarcinogenic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Chemoprevention; Delayed-Action Preparations; Drug Carriers; Drug Implants; Drug Liberation; Excipients; Fenretinide; Humans; Lactic Acid; Male; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Recurrence, Local; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats, Sprague-Dawley; Solubility; Xenograft Model Antitumor Assays

Over one third of patients who have undergone oral squamous cell carcinoma (OSCC) surgical resections develop life-threatening and often untreatable recurrences. A variety of drugs, intended for management of recurrent or disseminated cancers, were designed to exploit cancer cells' reliance upon overexpressed receptors and gratuitous signaling. Despite their conceptual promise, clinical trials showed these agents lacked efficacy and were often toxic. These findings are consistent with evasion of pathway-targeted treatments via extensive signaling redundancies and compensatory mechanisms common to cancers. Optimal secondary OSCC chemoprevention requires long-term efficacy with multifaceted, nontoxic agents. Accordingly, this study evaluated the abilities of three complementary chemopreventives, that is, the vitamin A derivative fenretinide (4-HPR, induces apoptosis and differentiation, inhibits signaling proteins, and invasion), the estrogen metabolite 2-methoxyestradiol (2-ME, apoptosis-inducing, antiangiogenic), and the humanized mAb to the IL6R receptor tocilizumab (TOC, reduces IL6 signaling) to suppress OSCC gratuitous signaling and tumorigenesis. Modeling studies demonstrated 4-HPR's high-affinity binding at STAT3's dimerization site and c-Abl and c-Src ATP-binding kinase sites. Although individual agents suppressed cancer-promoting pathways including STAT3 phosphorylation, STAT3-DNA binding, and production of the trans-signaling enabling sIL6R, maximal chemopreventive effects were observed with agent combinations. OSCC tumor xenograft studies showed that locally delivered TOC, TOC+4-HPR, and TOC+4-HPR+2-ME treatments all prevented significant tumor growth. Notably, the TOC+4-HPR+2-ME treatment resulted in the smallest overall increase in tumor volume. The selected agents use diverse mechanisms to disrupt tumorigenesis at multiple venues, that is, intracellular, tumor cell-ECM, and tumor microenvironment; beneficial qualities for secondary chemopreventives. Cancer Prev Res; 10(1); 76-88. ©2016 AACR.

Topics: 2-Methoxyestradiol; Animals; Antibodies, Monoclonal, Humanized; Anticarcinogenic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinogenesis; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Estradiol; Fenretinide; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Phenotype; Phosphorylation; Receptors, Interleukin-6; Signal Transduction; STAT3 Transcription Factor; Tumor Microenvironment; Xenograft Model Antitumor Assays

Combining an anticancer agent fenretinide (HPR) or C6-pyridinium ceramide (LCL29) with Foscan-mediated photodynamic therapy (FoscanPDT) is expected to augment anticancer benefits of each substance. We showed that treatment with FoscanPDT+HPR enhanced accumulation of C16-dihydroceramide, and that fumonisin B1 (FB), an inhibitor of ceramide synthase, counteracted caspase-3 activation and colony-forming ability of head and neck squamous cell carcinoma (HNSCC) cells. Because cancer cells appear to be more susceptible to increased levels of the endoplasmic reticulum (ER) stress than normal cells, herein we tested the hypothesis that FoscanPDT combined with HPR or LCL29 induces FB-sensitive ER stress-associated apoptosis that affects cell survival.. Using an HNSCC cell line, we determined: cell survival by clonogenic assay, caspase-3 activity by spectrofluorometry, the expression of the ER markers BiP and CHOP by quantitative real-time polymerase chain reaction and western immunoblotting, and sphingolipid levels by mass spectrometry.. Similar to HPR+FoscanPDT, LCL29+FoscanPDT induced enhanced loss of clonogenicity and caspase-3 activation, that were both inhibited by FB. Our additional pharmacological evidence showed that the enhanced loss of clonogenicity after the combined treatments was singlet oxygen-, ER stress- and apoptosis-dependent. The combined treatments induced enhanced, FB-sensitive, up-regulation of BiP and CHOP, as well as enhanced accumulation of sphingolipids.. Our data suggest that enhanced clonogenic cell killing after the combined treatments is dependent on oxidative- and ER-stress, apoptosis, and FB-sensitive sphingolipid production, and should help develop more effective mechanism-based therapeutic strategies.

Topics: Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Ceramides; Combined Modality Therapy; Endoplasmic Reticulum Stress; Fenretinide; Fumonisins; Head and Neck Neoplasms; Humans; Mesoporphyrins; Photochemotherapy; Pyridinium Compounds; Radiation-Sensitizing Agents; Squamous Cell Carcinoma of Head and Neck

The membrane-associated protein, focal adhesion kinase (FAK), modulates cell-extracellular matrix interactions and also conveys prosurvival and proliferative signals. Notably, increased intraepithelial FAK levels accompany transformation of premalignant oral intraepithelial neoplasia (OIN) to oral squamous cell carcinoma (OSCC). OIN chemoprevention is a patient-centric, optimal strategy to prevent OSCC's comorbidities and mortality. The cancer chemopreventive and synthetic vitamin A derivative, fenretinide, has demonstrated protein-binding capacities, for example, mTOR- and retinol-binding protein interactions. These studies used a continuum of human oral keratinocytes (normal-HPV E6/E7-transduced-OSCC) to assess potential fenretinide-FAK drug protein interactions and functional consequences on cellular growth regulation and motility. Molecular modeling studies demonstrated that fenretinide has approximately 200-fold greater binding affinity relative to the natural ligand (ATP) at FAK's kinase domain. Fenretinide also shows intermediate binding at FAK's FERM domain and interacts at the ATP-binding site of the closest FAK analogue, PYK2. Fenretinide significantly suppressed proliferation via induction of apoptosis and G2-M cell-cycle blockade. Fenretinide-treated cells also demonstrated F-actin disruption, significant inhibition of both directed migration and invasion of a synthetic basement membrane, and decreased phosphorylation of growth-promoting kinases. A commercially available FAK inhibitor did not suppress cell invasion. Notably, although FAK's FERM domain directs cell invasion, FAK inhibitors target the kinase domain. In addition, FAK-specific siRNA-treated cells showed an intermediate cell migration capacity; data which suggest cocontribution of the established migrating-enhancing PYK2. Our data imply that fenretinide is uniquely capable of disrupting FAK's and PYK2's prosurvival and mobility-enhancing effects and further extend fenretinide's chemopreventive contributions beyond induction of apoptosis and differentiation.

Topics: Anticarcinogenic Agents; Carcinoma, Squamous Cell; Cell Movement; Chemoprevention; Extracellular Matrix; Fenretinide; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Keratinocytes; Mouth Mucosa; Mouth Neoplasms; Precancerous Conditions; Protein Binding; Signal Transduction; Tumor Cells, Cultured

Because photodynamic therapy (PDT) alone is not always effective as an anticancer treatment, PDT is combined with other anticancer agents for improved efficacy. The clinically-relevant fenretinide [N-(4-hydroxyphenyl) retinamide; 4HPR], was combined with the silicon phthalocyanine photosensitizer Pc4-mediated PDT to test for their potential to enhance killing of SCC17B cells, a clinically-relevant model of human head and neck squamous cell carcinoma. Because each of these treatments induces apoptosis and regulates the de novo sphingolipid (SL) biosynthesis pathway, the role of ceramide synthase, the pathway-associated enzyme, in PDT+4HPR-induced apoptotic cell death was determined using the ceramide synthase inhibitor fumonisin B1 (FB). PDT+4HPR enhanced loss of clonogenicity. zVAD-fmk, a pan-caspase inhibitor, and FB, protected cells from death post-PDT+4HPR. In contrast, the anti-apoptotic protein Bcl2 inhibitor ABT199 enhanced cell killing after PDT+4HPR. Combining PDT with 4HPR led to FB-sensitive, enhanced Bax associated with mitochondria and cytochrome c redistribution. Mass spectrometry data showed that the accumulation of C16-dihydroceramide, a precursor of ceramide in the de novo SL biosynthesis pathway, was enhanced after PDT+4HPR. Using quantitative confocal microscopy, we found that PDT+4HPR enhanced dihydroceramide/ceramide accumulation in the ER, which was inhibited by FB. The results suggest that SCC17B cells are sensitized to PDT by 4HPR via the de novo SL biosynthesis pathway and apoptosis, and imply potential clinical relevance of the combination for cancer treatment.

Topics: Anticarcinogenic Agents; Apoptosis; Carcinoma, Squamous Cell; Combined Modality Therapy; Fenretinide; Head and Neck Neoplasms; Humans; Microscopy, Confocal; Photochemotherapy; Spectrometry, Mass, Electrospray Ionization; Sphingolipids; Tumor Cells, Cultured; Tumor Stem Cell Assay

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) has shown potential as a chemopreventive and therapeutic agent. The ability of 4HPR to enhance production of reactive oxygen species (ROS) leading to apoptosis has been suggested as a possible mechanism underlying these effects. We explored the possibility that ROS induction by 4HPR involves the small GTPase Ras-related C3 botulinum toxin substrate (Rac), a regulatory subunit of the NADPH oxidase complex. Rac was activated in human head and neck squamous cell carcinoma (HNSCC) cells as early as 5 minutes following 4HPR exposure. Moreover, inhibition of Rac activity or silencing of its expression by RNA interference decreased ROS generation in human head and neck, lung, and cervical cancer cells and murine melanoma cells. In HNSCC UMSCC-22B cells, this decrease correlated with reduction in apoptosis induction by 4HPR. Expression of a constitutive active mutant Rac increased basal and 4HPR-induced ROS generation and poly(ADP-ribose) polymerase cleavage. In addition, the metastatic DM14 cells exhibited higher Rac activation following 4HPR treatment compared with the primary Tu167-C2 cells. Furthermore, the metastatic cancer cells tested exhibited higher ROS generation and growth inhibition due to 4HPR exposure compared with their primary cancer cell counterparts. These findings show a preferential susceptibility of metastatic cells to the proapoptotic retinoid 4HPR through Rac activation and support the use of ROS-inducing agents such as 4HPR against metastatic cancer cells.

Topics: Aminoquinolines; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Fenretinide; Head and Neck Neoplasms; Humans; Pyrimidines; rac GTP-Binding Proteins; Reactive Oxygen Species

N-(4-hydroxyphenyl)retinamide (4HPR), which has shown efficacy in cancer chemopreventionand therapy, induces the mitochondrial apoptosis pathway via increased generation of reactive oxygen species (ROS). ROS is also known to be able to induce an endoplasmic reticulum (ER) stress response, which can contribute to apoptosis but may also antagonize it. Therefore, we used human head and neck squamous cell carcinoma (HNSCC) cells to determine whether 4HPR affects ER stress. Different experimental approaches have indicated that 4HPR induces ER stress response: electron microscopy, which showed extensive ER dilation; splicing of the X-box binding protein 1 (XBP-1), a marker of unfolded protein response (UPR) activation; and quantitative real-time PCR and immunoblotting, which revealed the upregulation of several ER-stress associated mRNAs and proteins, including the chaperone heat shock protein HSPA1A. Most of these effects of 4HPR were abrogated by cotreatment of cells with the antioxidant 3-tert-butyl-4-hydroxyanisole (BHA) indicating that they were downstream of the increase in ROS. Furthermore, siRNA-mediated silencing and chemical inhibition of HSPA1A, which exerts either pro- or anti-apoptotic effects, decreased 4HPR-induced apoptosis. These results demonstrate that 4HPR induces ER stress and uncovered a pro-apoptotic role for HSPA1A in 4HPR-induced apoptosis.

Topics: Anticarcinogenic Agents; Antioxidants; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Caspases; Cytochromes c; DNA-Binding Proteins; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Fenretinide; Head and Neck Neoplasms; HSP70 Heat-Shock Proteins; Humans; Nuclear Proteins; Oxidative Stress; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Regulatory Factor X Transcription Factors; Reverse Transcriptase Polymerase Chain Reaction; RNA Splicing; RNA, Messenger; RNA, Small Interfering; Transcription Factors; Tumor Cells, Cultured; X-Box Binding Protein 1

N-(4-hydroxyphenyl)retinamide (4HPR), a synthetic retinoid effective in cancer chemoprevention and therapy, is thought to act via apoptosis induction resulting from increased reactive oxygen species (ROS) generation. As ROS can activate MAP kinases and protein kinase C (PKC), we examined the role of such enzymes in 4HPR-induced apoptosis in HNSCC UMSCC22B cells. 4HPR increased ROS level within 1 h and induced activation of caspase 3 and PARP cleavage within 24 h. Activation of MKK3/6 and MKK4, JNK, p38 and ERK was detected between 6 and 12 h, increased up to 24 h and preceded apoptosis. 4HPR-induced activation of these kinases was abrogated by the antioxidants BHA and vitamin C. SP600125, a JNK inhibitor, suppressed 4HPR-induced c-Jun phosphorylation, cytochrome c release from mitochondria and apoptosis. Suppression of JNK1 and JNK2 using siRNA decreased, whereas overexpression of wild type-JNK1 enhanced 4HPR-induced apoptosis. PD169316, a p38, inhibitor suppressed phosphorylation of Hsp27 and apoptosis. PD98059, an MEK1/2 inhibitor, also suppressed ERK1/2 activation and apoptosis induced by 4HPR. Likewise, PKC inhibitor GF109203X suppressed ERK and p38 phosphorylation and PARP cleavage. These data indicate that 4HPR-induced apoptosis is triggered by ROS increase, leading to the activation of the mitogen-activated protein serine/threonine kinases JNK, p38, PKC and ERK, and subsequent apoptosis.

Topics: Anticarcinogenic Agents; Antioxidants; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Caspases; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Fenretinide; Head and Neck Neoplasms; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinase C; Proto-Oncogene Proteins c-jun; Reactive Oxygen Species; Tumor Cells, Cultured

It has been demonstrated that the chemopreventive agent N-(4-hydroxyphenyl)retinamide (4-HPR) induces apoptotic cell death, but recent data has suggested that late stage/recurrent tumours lose their response to 4-HPR-induced cell death by mechanisms that are unknown. Our study investigated the ability of 4-HPR to induce cell death in keratinocyte cell lines that represent different stages of carcinogenesis and the role of TGF-beta signalling in the induction of cell death by 4-HPR. We show that treatment of the immortalised keratinocyte cell line HaCaT with 10(-5) M 4-HPR induced cell death by apoptosis and caused an accumulation of cells in the G0/G1 phase of the cell cycle. Using a genetically related series of human skin keratinocytes derived from HaCaT that reflect tumour progression and metastasis in vivo, we demonstrate that 4-HPR-induced cell death and apoptosis is attenuated in the more aggressive tumour cell lines but that a reduced level of response is retained. Response to TGF-beta-induced growth inhibition was also reduced in the more aggressive cell lines. Treatment of HaCaT cells with 4-HPR induced TGF-beta2 expression and an increase in the amount of active TGF-beta in the culture medium. The inhibition of TGF-beta signalling attenuated 4-HPR-induced apoptosis and both TGF-beta1 and TGF-beta2 potentiated 4-HPR-induced apoptosis and enhanced 4-HPR-induced growth inhibition. Our results demonstrate that loss of response to 4-HPR correlates with a loss of response to the growth inhibitory effects of TGF-beta and that adjuvant therapies that upregulate TGF-beta may enhance the chemopreventive effects of 4-HPR.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Northern; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Line; Cell Proliferation; Disease Progression; Dose-Response Relationship, Drug; Drug Synergism; Fenretinide; Flow Cytometry; Gene Expression; Humans; Keratinocytes; Signal Transduction; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

When multiple drugs are administered simultaneously, investigators are often interested in assessing whether the drug combinations are synergistic, additive, or antagonistic. Based on the Loewe additivity reference model, many existing response surface models require constant relative potency and some of them use a single parameter to capture synergy, additivity, or antagonism. However, the assumption of constant relative potency is too restrictive, and these models using a single parameter to capture drug interaction are inadequate to describe the phenomenon when synergy, additivity, and antagonism are interspersed in different regions of drug combinations. We propose a generalized response surface model with a function of doses instead of one single parameter to identify and quantify departure from additivity. The proposed model can incorporate varying relative potencies among multiple drugs as well. Examples and simulations are given to demonstrate that the proposed model is effective in capturing different patterns of drug interaction.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biometry; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Data Interpretation, Statistical; Dose-Response Relationship, Drug; Drug Antagonism; Drug Interactions; Drug Synergism; Fenretinide; Humans; Models, Biological; Models, Statistical; Piperidines; Pyridines

N-(4-hydroxyphenyl)retinamide (4HPR) is a synthetic retinoid that has shown efficacy in cancer chemoprevention and therapy possibly due to its ability to induce growth inhibition and apoptosis in cancer cells. Reactive oxygen species (ROS) have been implicated in mediating 4HPR-induced apoptosis in various cancer cells, including head and neck squamous cell carcinoma (HNSCC) cells. 4HPR increased the level of p67phox, which is a subunit of the NADPH oxidase complex that is involved in the generation of ROS. The increase in the level of p67phox protein may be a downstream effect of the activation of c-Jun N-terminal kinase (JNK) induced by 4HPR. Suppression of endogenous and 4HPR-induced levels of p67phox using small interfering RNA did not result in a change in ROS generation or apoptosis. These results suggest that p67phox is not a critical component or a limiting factor in the 4HPR-induced apoptosis pathway in HNSCC cells.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Enzyme Activation; Fenretinide; Head and Neck Neoplasms; Humans; JNK Mitogen-Activated Protein Kinases; NADPH Oxidases; Phosphoproteins; Reactive Oxygen Species; RNA, Small Interfering; Time Factors; Transfection

Several genetic alterations have been described in cervical cancers including: human papillomavirus (HPV) E6 and E7 oncoproteins, subtle sequence changes, alterations in chromosome number, chromosome translocations, and gene amplifications. This report focuses on establishing chromosome 9 polysomy as a cervical biomarker of chromosome instability and using it in a chemoprevention trial. Chromosomal instability is a feature of most human cancers and is probably an early event in the process.. We used 37 cervical cone specimens to validate chromosome 9 polysomy as a biomarker and then tested its modulation in a randomized clinical trial of 4-hydroxyphenylretinamide (4-HPR) in 39 patients with three blinded histopathologic reviews. No confounders were identified. In the present study, immunohistocytochemical analysis of Chromosome 9 polysomy was carried out and quantitatively measured.. The Cell Index, the ratio of the number of total chromosome 9 copies to the total number of ells, increases significantly in archival samples as the cervix changes from normal to CIN to invasive cancer. In the chemoprevention trial, chromosome 9 polysomy was used as a biomarker and supported the histological analysis showing that 4-HPR impaired the natural regression response.. Chromosome 9 polysomy appears to be a marker of genetic instability that can be used in chemoprevention trials as a surrogate endpoint biomarker. In this randomized trial of 4-HPR, the chromosome 9 polysomy measurements supported the clinical histopathologic reading in a quantitative manner suggesting that 4-HPR at 200 mg/day may have been inhibiting the regression seen in the placebo arm by inducing genetic instability.

Topics: Aneuploidy; Anticarcinogenic Agents; Carcinoma, Squamous Cell; Chromosomes, Human, Pair 9; Female; Fenretinide; Humans; Placebos; Randomized Controlled Trials as Topic; Reproducibility of Results; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

To explore whether the mechanism of action of 4-hydroxyphenylretinamide (4-HPR, fenretidine), a synthetic retinoid, involves the functional activation of the nuclear hormone receptor class known as PPARs (peroxisome proliferator-activated receptors). Also, to examine whether anti-proliferative effects of this agent in head and neck cancer cells occur at biologically relevant concentrations.. CA 9-22, NA, and UM SCC 11B cells were treated with 4-HPR during their log phase growth and functional activation of PPAR gamma was evaluated by plate luminometry. Cellular proliferation was analyzed by standard MTT cell proliferation assays and cell counting. Student's t tests were performed for all experiments.. Significant dose-dependent increases in PPAR gamma activation occurred in response to 4-HPR treatment. Proliferation was significantly inhibited by 4-HPR in a dose-dependent manner as judged by MTT and cell counting assays. These effects occurred at equimolar concentrations in both types of experiments within a range of clinically achievable doses (1-4 microM) of 4-HPR.. 4-HPR can functionally activate PPAR gamma at clinically achievable doses. Decreased cancer cell proliferation secondary to PPAR gamma activation has been observed in other malignancies as well as upper aerodigestive cancer. PPAR gamma activation by 4-HPR represents another potential anti-cancer mechanism of action for this drug.. PPAR gamma activation represents a novel target for anti-cancer therapy for head and neck cancer and the current level of clinical toxicity of 4-HPR would be judged acceptable to utilize this agent alone or in combination chemotherapy.

Topics: Carcinoma, Squamous Cell; Cell Death; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Female; Fenretinide; Humans; Male; Mouth Neoplasms; Peroxisome Proliferator-Activated Receptors; Probability; Sampling Studies; Sensitivity and Specificity; Tumor Cells, Cultured

Retinoids have shown clinical efficacy in cancer chemoprevention and therapy presumably by modulating the growth, differentiation, and apoptosis of normal, premalignant, and malignant cells. To better understand the mechanisms by which retinoids exert their effects, we used a high-throughput Western blotting method (Becton-Dickinson PowerBlot) to evaluate changes in the levels of cellular signaling proteins in head and neck squamous cell carcinoma cells treated with the cytostatic all-trans-retinoic acid or with the proapoptotic retinoids 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid or N-(4-hydroxyphenyl)retinamide. Treatments of the head and neck squamous cell carcinoma cells with these retinoids for 24 h resulted in increased levels of 14, 22, and 22 proteins and decreased levels of 5, 10, and 7 proteins, respectively. The changes in the levels of the following proteins were confirmed by conventional western immunoblotting: all-trans-retinoic acid increased ELF3, topoisomerase II alpha, RB2/p130, RIG-G, and EMAPII and decreased MEF2D and cathepsin L. N-(4-Hydroxyphenyl)retinamide up-regulated ELF3, c-Jun, Rb2/p130, JAK1, p67phox, Grb2, O(6)-methylguanine-DNA methyltransferase, and Ercc-1. 6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid increased Rb2/p130, c-Jun, Sp1, Sin, and tomosyn and decreased cathepsin L, Mre11, and topoisomerase II alpha. Some of these proteins were also modulated by these retinoids in other human cancer cell lines. A subset of the proteins were modulated similarly by the different retinoids, whereas changes in other proteins were unique for each retinoid. These results suggest that the mechanisms by which these retinoids modulate proteins are distinct but may overlap. Some of the retinoid-modulated proteins identified in this study may be novel candidates for mediating different responses to retinoids.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Fenretinide; Head and Neck Neoplasms; Humans; Neoplasm Proteins; Retinoids; Tretinoin

Squamous cell carcinoma (SCC) is the most prevalent form of epithelial cancer. SCC results when normal epithelial cells undergo multiple neoplastic changes that culminate in the evolution of an invasive cancer. Retinoids are commonly used as chemopreventive and treatment agents in skin cancer; however, SCC progression is accompanied by a gradual loss of retinoid responsiveness. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has shown promising anti-neoplastic activity in a variety of tumor cells, including those that are resistant to all-trans retinoic acid (t-RA). We investigated the effect of HPR on growth and apoptosis of squamous cells at different stages of carcinogenesis. We then determined if retinoic acid receptor (RAR) overexpression affected the outcome of HPR treatment. To model SCC malignant progression, we used a panel of murine keratinocytes representing different stages of squamous cell carcinogenesis. This panel consisted of primary keratinocytes, SP1 and 308 papilloma cell lines, the PAM-212 squamous carcinoma cell line, and the spindle I7 cell line. With the exception of the primary keratinocytes, all cells were unresponsive to t-RA treatment. Pharmacological concentrations of HPR were non-cytotoxic to all keratinocytes tested and HPR sensitivity was stage-dependent, with the papilloma cell lines being the most sensitive, and the spindle cells being the most resistant. Overexpression of RARgamma in SP1 papilloma cells enhanced growth suppression and apoptosis induction by HPR. HPR-induced growth suppression was accompanied by a simultaneous block in the G(1) phase of the cell cycle in RAR-transduced and control SP1 cells and differential regulation of cell cycle and apoptotic mediators.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzimidazoles; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Fenretinide; G1 Phase; Keratinocytes; Mice; Mice, Inbred BALB C; Neoplasm Staging; Nevus, Spindle Cell; Papilloma; Propidium; Receptors, Retinoic Acid; Ribonucleases; Tretinoin

The retinoid 4-(N-hydroxyphenyl)retinamide (4HPR, fenretinide) has both growth inhibitory and apoptosis-inducing effects on a number of cancer cell lines in vitro and in vivo and has been entered into a number of oncological trials. However, little is known about its mechanism(s) of action or its effects on normal cells such as fibroblasts. In this study, the effects of fenretinide on both epidermoid carcinoma cells of vulva (cell line A431) and normal human dermal fibroblasts, both as monolayers and also grown in 3D cell culture systems, have been investigated. The 3D cell culture system contained normal human fibroblasts embedded in a type I collagen gel with the carcinoma cells seeded on top of the collagen gel, which mimics the epidermoid carcinoma. Fenretinide significantly inhibited the rate of DNA synthesis of carcinoma cells, while there was little effect on fibroblasts on monolayers, at 10(-6)-10(-5) M, which are clinically attainable doses. Fenretinide at 5 x 10(-6) M induced apoptosis characterised by cell shrinkage, membrane blebbing, nuclear condensation and/or fragmentation, and cell detachment in carcinoma cells, but not fibroblasts from monolayers. Fenretinide also reduced the viability of carcinoma cells in the 3D cell culture system without affecting fibroblasts. These data show that fenretinide may preferentially induce apoptosis in epidermoid carcinoma cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Culture Techniques; Cell Survival; Collagen; DNA Fragmentation; Fenretinide; Fibroblasts; Humans; Nucleosomes; Rats; Reference Values; Skin; Tumor Cells, Cultured

The inhibitory effects of N-(4-hydroxyphenyl)retinamide (4HPR) on the process of carcinogenesis are not fully understood and may result from its ability to induce apoptosis in transformed cells. This study investigated the apoptotic properties of 4HPR in four human cutaneous squamous cell carcinoma cell lines. Apoptosis induction, detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling method, occurred in a dose- and time-dependent fashion after treatment with 4HPR. 4HPR promoted reactive oxygen species (ROS) determined by oxidation of 2',7'-dichlorofluorescin. 4HPR-induced ROS, and apoptosis could be inhibited by L-ascorbic acid. Rhodamine 123 retention revealed that 4HPR treatment promoted a gradual dissipation of mitochondrial inner transmembrane potential, and this could be inhibited by L-ascorbic acid, implying that mitochondrial permeability transition was involved in apoptosis induction. Cyclosporin A and bongkrekic acid inhibited dissipation of mitochondrial inner transmembrane potential, ROS production, and DNA fragmentation after exposure to 4HPR, demonstrating that mitochondrial permeability transition was a central coordinating feature of 4HPR-induced apoptosis.

Topics: Anticarcinogenic Agents; Apoptosis; Ascorbic Acid; Carcinoma, Squamous Cell; Cell Division; Cyclosporine; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Fenretinide; Fluoresceins; Fluorescent Dyes; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Oxidation-Reduction; Permeability; Reactive Oxygen Species; Rhodamine 123; Skin Neoplasms; Tumor Cells, Cultured

The induction of apoptosis by the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) has been documented in vitro in various cancer types. A role for reactive oxygen species (ROS) in apoptosis induced by 4HPR in some cancer cells has been demonstrated recently. We studied five different human head and neck and five lung cancer cell lines to determine whether the ROS play a general role in 4HPR-induced apoptosis. We found that 4HPR induced apoptosis in all of the cell lines; however, this effect was blocked by antioxidants in only 2 of the 10 cell lines. 4HPR induced a greater than 4-fold increase in the generation of intracellular ROS in these two cell lines compared with a much lower effect in other cell lines. Furthermore, these two cell lines were most sensitive to the induction of apoptosis by 4HPR. The level of the cellular antioxidant thiol and superoxide dismutase activity were relatively lower in cells, which responded to 4HPR with a high level of ROS generation. These results indicate that although ROS can mediate 4HPR-induced apoptosis in some cells, which may have a low endogenous cellular antioxidant levels, other mechanisms exist for 4HPR-induced apoptosis. One such mechanism may involve retinoic acid receptors (RARs) because an RAR antagonist was able to block partially 4HPR-induced apoptosis. In conclusion, 4HPR-induced apoptosis involves at least three different mechanisms, which are complex and can overlap in the same cell line: (a) one mechanism involving 4HPR-induced ROS; (b) one involving RARs; and (c) at least one that does not involve ROS or RARs and remains unclear.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; Ascorbic Acid; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Fenretinide; Head and Neck Neoplasms; Humans; Lung Neoplasms; Naphthalenes; Neoplasm Proteins; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Receptors, Retinoic Acid; Sulfhydryl Compounds; Superoxide Dismutase; Tumor Cells, Cultured

N-(4-Hydroxyphenyl)retinamide (4HPR) is currently used in cancer prevention and therapy trials. It is thought that its effects result from induction of apoptosis. 4HPR-induced apoptosis in human cervical carcinoma C33A cells involves enhanced generation of reactive oxygen species (ROS). In this study we explored the mechanism by which 4HPR increases ROS and induces apoptosis in these cells. 4HPR induced cytochrome c release from mitochondria to cytoplasm, activated caspase-3, and caused a membrane permeability transition (MPT). All these 4HPR's effects, as well as the induction of apoptosis, were inhibited by antioxidants, which decrease ROS. Thenoyltrifluoroacetone, a mitochondrial respiratory chain (MRC) complex II inhibitor, and carbonylcyanide m-chlorophenyl hydrazone, which uncouples electron transfer and ATP synthesis and inhibits ROS generation by MRC, inhibited 4HPR-induced ROS generation very effectively. Rotenone, an MRC complex I inhibitor was less effective and azide, an MRC complex IV inhibitor, exhibited a marginal effect. In contrast, antimycin A, an MRC complex III inhibitor, enhanced 4HPR-induced ROS generation. These findings suggest that 4HPR enhances ROS generation by affecting a target between complex II and complex III, presumably coenzyme Q. This effect is followed by release of cytochrome c, increased caspase-3 activity, induction of MPT and eventual DNA fragmentation and cell death.

Topics: Anticarcinogenic Agents; Antimycin A; Antineoplastic Agents; Antioxidants; Apoptosis; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carcinoma, Squamous Cell; Caspase 3; Caspases; Cytochrome c Group; Electron Transport; Electron Transport Complex I; Electron Transport Complex II; Electron Transport Complex III; Enzyme Inhibitors; Female; Fenretinide; Humans; Mitochondria; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Neoplasm Proteins; Oxidoreductases; Reactive Oxygen Species; Rotenone; Sodium Azide; Succinate Dehydrogenase; Thenoyltrifluoroacetone; Tumor Cells, Cultured; Uncoupling Agents; Uterine Cervical Neoplasms

Squamous cell carcinoma of the head and neck (HNSCC) has a high incidence of recurrence and associated second primary malignancy. The retinoid 13-cis-retinoic acid has been shown to be effective as both a chemopreventive and chemotherapeutic agent for HNSCC, but often with treatment-limiting toxicity. The synthetic retinoid fenretinide (N-(4-hydroxyphenyl)retinamide) (HPR) has significant antiproliferative activity against a number of animal and human malignancies and has been used in clinical trials as a chemopreventive agent in patients with breast and prostate cancer and oral leukoplakia. HPR has been shown to have a toxicity profile lower than that for other retinoids used in clinical trials.. The aim of this study was to investigate the effect of HPR on the growth of HNSCC cell lines in vitro.. Four HNSCC cell lines (JHU-011-SCC, JHU-020-SCC, JHU-022-SCC, and FaDu) were treated with a range of concentrations of HPR for various times. After HPR exposure, cell viability was determined by tetrazolium dye (MTT) colorimetric assay, comparing cell survival with that of untreated control cells. HPR-induced apoptosis was determined by flow-cytometric deoxyribonucleic acid cell-cycle analysis, ultrastructural analysis with electron microscopy, and deoxyribonucleic acid fragmentation detected by gel electrophoresis.. HPR caused significant growth inhibition in three of the four HNSCC cell lines in a dose- and time-dependent fashion. In two cell lines (JHU-011-SCC, JHU-020-SCC) a significant antiproliferative effect was achieved between 1 and 2.5 micromol/L HPR after 72 hours of treatment. By deoxyribonucleic acid cell-cycle analysis, electron microscopy, and gel electrophoresis, HPR was shown to induce apoptosis in the JHU-011-SCC and JHU-020-SCC cell lines, but not in the FaDu cell line, which was insensitive to the growth inhibitory effect of HPR.. This study has demonstrated that HPR reduces cell viability in HNSCC cells in vitro at clinically relevant doses, with the growth inhibition occurring through the induction of apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Division; Electrophoresis, Agar Gel; Fenretinide; Flow Cytometry; Head and Neck Neoplasms; Humans; In Vitro Techniques; Laryngeal Neoplasms; Microscopy, Electron; Tumor Cells, Cultured

Retinoids are vitamin A analogs that regulate growth and differentiation of squamous epithelial cells in vitro and in vivo. We examined the effects of retinoic acid (RA) and N-(4-hydroxy-phenyl) retinamide (HPR) on the growth properties of esophageal squamous carcinoma cell lines, and found that both RA and HPR induce morphological changes and inhibit growth. Immunofluorescence studies suggest alterations in keratins as a result of treatment with RA or HPR. In order to determine underlying molecular mechanisms, we found that RA or HPR did not induce arrest of cells in the G1 phase nor did treated cells undergo apoptosis. However, RA and HPR treatment did result in the downregulation of epidermal growth factor receptor (EGFR) which is known to bind key proproliferative ligands, such as epidermal growth factor and transforming growth factor alpha.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Division; Cell Size; ErbB Receptors; Esophageal Neoplasms; Fenretinide; Flow Cytometry; Humans; Keratins; Time Factors; Tretinoin; Tumor Cells, Cultured

Both retinoic acid (RA) and the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) have shown efficacy in head and neck cancer chemoprevention trials. To compare their activity and mechanism of action, the 1483 oral head and neck squamous cell carcinoma (HNSCC) cell line was grown in organotypic culture, an in vitro system that allows cellular stratification and simulates carcinoma in situ, and was exposed to 10 micromol/L of either RA or 4HPR. Extensive apoptosis, as evidenced by in situ deoxyribonucleic acid end-labeling, occurred in 4HPR-treated cultures after 9 days, with >80% cell loss (P< .001). In contrast, the growth of cultures treated with RA was inhibited by only 32%, with no evidence of apoptosis. Because 4HPR has low systemic toxicity and is a potent inducer of apoptosis in HNSCC cells, its role in chemoprevention of head and neck cancers, including cancers that are resistant to RA-induction therapy, warrants further investigation.

Topics: Apoptosis; Carcinoma, Squamous Cell; Drug Screening Assays, Antitumor; Fenretinide; Head and Neck Neoplasms; Humans; Tretinoin; Tumor Cells, Cultured

Retinoids have been shown to act as cytostatic agents against a variety of tumor cell types, including squamous carcinoma cells. Recently it was reported that certain retinoids can induce apoptosis as well. Because we are investigating the potential of retinoids in chemoprevention and therapy for head and neck premalignant and malignant lesions, we compared the effects of all-trans-retinoic acid (ATRA) and N-(4-hydroxyphenyl)retinamide (4HPR) on seven human head and neck squamous cell carcinoma cell lines (17A, 17B, 22A, 22B, 38, SqCC/Y1, and 1483). Six of the seven cell lines showed dramatic morphological changes after treatment with 10 micrometer 4HPR, whereas no such changes were induced by 10 micrometer ATRA. To determine whether these retinoids can induce apoptosis, we analyzed both detached and attached cells after 2, 5, and 7 days of treatment for evidence of DNA fragmentation by DNA electrophoresis on agarose gels. In five of the seven cell lines, a DNA ladder was observed after treatment with 10 micrometer 4HPR for 5 or 7 days, whereas treatment with ATRA resulted in a less pronounced effect. In 17B cells, a clear DNA ladder was observed as early as 2 days after treatment with 4HPR; however, neither ATRA nor 9-cis-retinoic acid was as effective. In addition, morphological changes associated with apoptotic cell death, such as chromatin condensation and nuclear segmentation, were observed by propidium iodide staining and by electron microscopic analysis after 4HPR treatment. These results demonstrate that 4HPR causes apoptosis in several head and neck squamous cell carcinoma cell lines and that it is more potent in this effect than ATRA.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Fenretinide; Head and Neck Neoplasms; Humans; Tretinoin; Tumor Cells, Cultured

Several studies have correlated the excessive production of prostaglandins (PGs) with tumor promotion and the suppression of the immune response. Inhibition of PGs by pharmacological agents has been demonstrated to enhance immunocompetence, and to suppress growth of tumors in animals and humans. In this study we examined the effect of retinol (I), all-trans-retinoic acid (II), N-(4-Hydroxyphenyl) retinamide (N-4-HPR) (III), canthaxanthin (CTX) (IV), and beta-carotene (beta-CT) (V) on the bioconversion of 14C-arachidonic acid (AA) to PGE2 by squamous carcinoma cells of the tongue, SCC-25. Agents (I), (II), (III), (IV) inhibited while (V) stimulated PGE2 formation in a dose related manner. N-4-HPR was the most potent inhibitor of PGE2 synthesis. The data suggest that certain retinoids and carotenoids have the potential of inhibition of PG synthesis by oral squamous carcinoma cells. Inhibitory effects such as those described here and antioxidant properties might in part contribute to the antiinflammatory and anticarcinogenic activity of retinoids in vivo.

Topics: beta Carotene; Canthaxanthin; Carcinoma, Squamous Cell; Carotenoids; Chromatography, Thin Layer; Fenretinide; Humans; Indomethacin; Prostaglandins; Retinoids; Tongue Neoplasms; Tretinoin; Tumor Cells, Cultured; Vitamin A