dactolisib and Colonic-Neoplasms

Colon cancer is a top lethal cancer in man and women worldwide and drug resistance is the major cause of cancer-related death. Combinational therapy and drug delivery with nanoparticles have been shown to effectively overcome drug resistance in many cancers. We previously reported that nanoemulsion (NE) loaded paclitaxel (PTX) and BEZ235 could synergistically inhibit colon cancer cell growth.. To investigate whether NE loaded PTX and BEZ235 can overcome drug resistance and synergistically inhibit drug-resistant colon cancer cell growth in vitro and in vivo.. The in vitro treatment effect on cell viability was assayed using CCK8 kit, cell morphological change was detected by β-tubulin immunofluorescence staining, drug resistance-related proteins were analyzed by Western blotting, and in vivo tumor growth test was performed in nude mice xeno-transplanted with 2 drug-resistant colon cancer cell lines HCT116-LOHP and HT29-DDP.. Both cell lines were sensitive to PTX but relatively insensitive to BEZ235. PTX combined with BEZ235 synergistically inhibited the proliferation of both cell lines. Nanoemulsion loaded PTX (NE-PTX) reduced the IC50 of PTX to approximately 2/5 of free PTX, indicating a high inhibitory efficacy of NE-PTX. When NE-PTX combined with a low concentration of BEZ235 (50 nM), the IC50 was decreased to approximately 2/3 of free PTX. Moreover, NE-PTX+BEZ235 treatment increased apoptosis, decreased Pgp and ABCC1 expression, and reduced tumor weights compared to the single drug treatment and the control group. These results suggest that nanoemulsion loaded PTX+BEZ235 can overcome drug resistance and improve the inhibitory effect on cancer cell proliferation and tumor growth.. Our study thus provides a possible new approach to treat colon cancer patients with drug resistance.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Count; Cell Line, Tumor; Cell Proliferation; Cell Shape; Colonic Neoplasms; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Emulsions; Female; Humans; Imidazoles; Inhibitory Concentration 50; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Neoplasm Proteins; Paclitaxel; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

To explore whether GOLPH3 regulated oxaliplatin (L-OHP) resistance of colon cancer cells via PI3K/AKT/mTOR pathway.. HCT116/L-OHP cells were divided into Blank, Control/GOLPH3 shRNA, BEZ235 (a PI3K/AKT/mTOR inhibitor), and GOLPH3 + BEZ235 groups followed by the detection with MTT, soft agar colony formation, flow cytometry and TUNEL assays. Mice bearing HCT116/L-OHP xenografts were randomized into Control, L-OHP, NC/GOLPH3 shRNA, L-OHP + NC/GOLPH3 shRNA groups. The expressions of Ki67, Caspase-3, and PI3K/AKT/mTOR pathway proteins were examined by immunohistochemistry.. HCT116/L-OHP cells had increased GOLPH3 expression compared to HCT116 cells, which positively regulated PI3K/AKT/mTOR pathway in HCT116/L-OHP cells. BEZ235 declined IC50 of HCT116/L-OHP cells to L-OHP, decreased the expressions of ABCB1, ABCC1, ABCG2, ATP7A, ATP7B, MATE1, p-gp, MRP1 and BCRP, induced cell apoptosis, reduced cell proliferation, and arrested cells at G0/G1, which was reversed by GOLPH3 overexpression. L-OHP and GOLPH3 shRNA decreased tumor volume and reduced expression of Ki67 in tumor tissues with the increased Caspase-3. Meanwhile, the combined treatment had the better treatment effect.. GOLPH3 inhibition reduced proliferation and promoted apoptosis of HCT116/L-OHP cells, and also reversed the L-OHP resistance of HCT116/L-OHP, which may be associated with the suppression of P13K/AKT/mTOR pathway.

Topics: Animals; Apoptosis; Cell Cycle; Colonic Neoplasms; Drug Resistance, Neoplasm; HCT116 Cells; Humans; Imidazoles; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Oxaliplatin; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quinolines; RNA, Small Interfering; Signal Transduction; TOR Serine-Threonine Kinases

Programmed cell death ligand 1 (PD-L1) is an important immune-inhibitory protein expressed on cancer cells to mediate cancer escape through interaction with PD-1 expressed on activated T lymphocytes (T cells). Previously, we reported that colon and breast cancer stem cells (CSCs) expressed much higher levels of PD-L1 than their parental cells, suggesting they will be more resistant to immune attack.. We investigated the underlining mechanism of PD-L1 increase in colon CSCs, with a special focus on the effect of insulin and epithelial growth factor (EGF), the two fundamental components to sustain the metabolism and stemness in the culture of CSCs.. We found that insulin increased the total and surface PD-L1 levels through PI3K/Akt/mTOR pathway as the increase could be inhibited by the dual inhibitor of the pathway, BEZ235. EGF didn't affect the total PD-L1 levels of CSCs but increased the cell surface protein levels by flow cytometry analysis, indicating EGF promotes the transport of PD-L1 to the cell surface. Blocking cell surface PD-L1 with a specific antibody resulted in a significant reduction of tumour sphere formation but didn't interfere with the sphere growth, suggesting that cell surface PD-L1 may act as an adhering molecule for CSCs.. Apart from the essential roles in metabolism and stemness, insulin and EGF involve in up-regulation of PD-L1 expression in colon CSCs, therefore the inhibition of insulin and EGF/EGFR pathways can be considered for cancer immunotherapy or combined with PD-1/PD-L1 antibody-based cancer immunotherapy to eliminate CSCs.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; B7-H1 Antigen; Cell Membrane; Colonic Neoplasms; Cytoplasm; Epidermal Growth Factor; HT29 Cells; Humans; Imidazoles; Immunotherapy; Insulin; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Up-Regulation

The phosphoinositide 3-kinase (PI3K) signaling pathway is critical for multiple important cellular functions, and is one of the most commonly altered pathways in human cancers. We previously developed a mouse model in which colon cancers were initiated by a dominant active PI3K p110-p85 fusion protein. In that model, well-differentiated mucinous adenocarcinomas developed within the colon and initiated through a non-canonical mechanism that is not dependent on WNT signaling. To assess the potential relevance of PI3K mutations in human cancers, we sought to determine if one of the common mutations in the human disease could also initiate similar colon cancers. Mice were generated expressing the Pik3caH1047R mutation, the analog of one of three human hotspot mutations in this gene. Mice expressing a constitutively active PI3K, as a result of this mutation, develop invasive adenocarcinomas strikingly similar to invasive adenocarcinomas found in human colon cancers. These tumors form without a polypoid intermediary and also lack nuclear CTNNB1 (β-catenin), indicating a non-canonical mechanism of tumor initiation mediated by the PI3K pathway. These cancers are sensitive to dual PI3K/mTOR inhibition indicating dependence on the PI3K pathway. The tumor tissue remaining after treatment demonstrated reduction in cellular proliferation and inhibition of PI3K signaling.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Carcinogenesis; Class I Phosphatidylinositol 3-Kinases; Colonic Neoplasms; Drug Screening Assays, Antitumor; Female; Genetic Association Studies; Genetic Predisposition to Disease; Imidazoles; Male; Mice, Inbred C57BL; Mice, Transgenic; Mutation, Missense; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; TOR Serine-Threonine Kinases; Tumor Burden

Colon cancer is the third most common cancer in the world, with drug resistance and metastasis being the major challenges to effective treatments. To overcome this, combination therapy with different chemotherapeutics is a common practice. In this study, we demonstrated that paclitaxel (PTX) together with BEZ235 exhibited a synergetic inhibition effect on colon cancer cell growth. Furthermore, nanoemulsion (NE)-loaded PTX and BEZ235 were more effective than the free drug, and a combination treatment of both NE drugs increased the efficiency of the treatments. BEZ235 pretreatment before adding PTX sensitized the cancer cells further, suggesting a synergistic inhibition effect through the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin pathway. The 50% inhibitory concentrations for BEZ235 were 127.1 nM and 145.0 nM and for PTX 9.7 nM and 9.5 nM for HCT-116 and HT-29 cells, respectively. When loaded with NE the 50% inhibitory concentrations for BEZ235 decreased to 52.6 nM and 55.6 nM and for PTX to 1.9 nM and 2.3 nM for HCT-116 and HT-29 cells, respectively. Combination treatment with 10 nM NE-BEZ235 and 0.6 nM and 1.78 nM NE-PTX could kill 50% of HCT-116 and HT-29, respectively. The cell death caused by the treatment was through apoptotic cell death, which coincided with decreased expression of anti-apoptotic protein B-cell lymphoma 2. Our data indicate that the combination therapy of PTX with the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin dual inhibitor BEZ235 using NE delivery may hold promise for a more effective approach for colon cancer treatment.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Colonic Neoplasms; Drug Delivery Systems; Emulsions; HCT116 Cells; HT29 Cells; Humans; Imidazoles; Nanocomposites; Paclitaxel; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; TOR Serine-Threonine Kinases

Colon cancer is one of the most common cancers worldwide with high mortality. A major issue in colon cancer treatment is drug-resistance and metastasis that have been ascribed to the cancer stem cells. In this study, colon cancer stem cells were isolated through sphere culture and verified with the cancer stem cell markers CD133, CD44, and CD24. It was demonstrated that the PI3K/Akt/mTOR signalling pathway was highly activated in the colon cancer stem cells and that inhibition of the PI3K/Akt/mTOR pathway by the inhibitor BEZ235 suppressed the colon cancer stem cell proliferation with reduced stemness indicated by CD133 and Lgr5 expressions. Treatment with insulin as a known activator of the PI3K/Akt pathway increased CD133 expression and decreased the effects of BEZ235 on colon cancer proliferation and survival. The data presented here collectively suggest that the PI3K/Akt/mTOR pathway underpins the stemness of colon cancer stem cells and BEZ235 is potentially a good drug candidate for treatment of colon cancer drug resistance and metastasis.

Topics: Antineoplastic Agents; Cell Survival; Colonic Neoplasms; Drug Resistance, Neoplasm; HCT116 Cells; Humans; Imidazoles; Insulin; Neoplasm Metastasis; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases

Apoptosis, or programmed cell death, can be leveraged as a surrogate measure of response to therapeutic interventions in medicine. Cysteine aspartic acid-specific proteases, or caspases, are essential determinants of apoptosis signaling cascades and represent promising targets for molecular imaging. Here, we report development and in vivo validation of [(18)F]4-fluorobenzylcarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone ([(18)F]FB-VAD-FMK), a novel peptide-based molecular probe suitable for quantification of caspase activity in vivo using positron emission tomography (PET).. Supported by molecular modeling studies and subsequent in vitro assays suggesting probe feasibility, the labeled pan-caspase inhibitory peptide, [(18)F]FB-VAD-FMK, was produced in high radiochemical yield and purity using a simple two-step, radiofluorination. The biodistribution of [(18)F]FB-VAD-FMK in normal tissue and its efficacy to predict response to molecularly targeted therapy in tumors was evaluated using microPET imaging of mouse models of human colorectal cancer.. Accumulation of [(18)F]FB-VAD-FMK was found to agree with elevated caspase-3 activity in response to Aurora B kinase inhibition as well as a multidrug regimen that combined an inhibitor of mutant BRAF and a dual PI3K/mTOR inhibitor in (V600E)BRAF colon cancer. In the latter setting, [(18)F]FB-VAD-FMK PET was also elevated in the tumors of cohorts that exhibited reduction in size.. These studies illuminate [(18)F]FB-VAD-FMK as a promising PET imaging probe to detect apoptosis in tumors and as a novel, potentially translatable biomarker for predicting response to personalized medicine.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Colonic Neoplasms; Colorectal Neoplasms; Female; Fluorine Radioisotopes; Fluorobenzenes; Humans; Imidazoles; Immunoblotting; Immunohistochemistry; Indoles; Mice, Inbred C57BL; Mice, Nude; Organophosphates; Peptides; Positron-Emission Tomography; Protein Kinase Inhibitors; Quinazolines; Quinolines; Radiopharmaceuticals; Sulfonamides; Tissue Distribution; Xenograft Model Antitumor Assays

Colon cancer is the third most common cancer and the third largest cause of cancer-related death. Fluorouracil (5-FU) is the front-line chemotherapeutic agent for colon cancer. However, its response rate is less than 60%, even in combination with other chemotherapeutic agents. The side effects of 5-FU also limit its application. Nanoparticles have been used to deliver 5-FU, to increase its effectiveness and reduce side effects. Another common approach for colon cancer treatment is targeted therapy against the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. A recently-invented inhibitor of this pathway, BEZ-235, has been tested in several clinical trials and has shown effectiveness and low side effects. Thus, it is a very promising drug for colon cancer treatment. The combination of these two drugs, especially nanoparticle-packed 5-FU and BEZ-235, has not been studied. In the present study, we demonstrated that nanoparticles of layered double hydroxide (LDH) loaded with 5-FU were more effective than a free drug at inhibiting colon cancer cell growth, and that a combination treatment with BEZ-235 further increased the sensitivity of colon cancer cells to the treatment of LDH-packed 5-FU (LDH-5-FU). BEZ-235 alone can decrease colon cancer HCT-116 cell viability to 46% of the control, and the addition of LDH-5-FU produced a greater effect, reducing cell survival to 8% of the control. Our data indicate that the combination therapy of nanodelivered 5-FU with a PI3K/Akt inhibitor, BEZ-235, may promise a more effective approach for colon cancer treatment.

Topics: Apoptosis; Cell Cycle; Cell Proliferation; Colonic Neoplasms; HCT116 Cells; Humans; Hydroxides; Imidazoles; Nanoparticles; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases

Regrowth of tumour cells between cycles of chemotherapy is a significant clinical problem. Treatment strategies where antiproliferative agents are used to inhibit tumour regrowth between chemotherapy cycles are attractive, but such strategies are difficult to test using conventional monolayer culture systems.. We used the in vitro tumour spheroid model to study regrowth of 3-D colon carcinoma tissue after cytotoxic therapy. Colon carcinoma cells with wild-type or mutant phosphatidyl inositol 3-kinase catalytic subunit (PI3KCA) or KRAS alleles were allowed to form multicellular spheroids and the effects of different pharmacological compounds were studied after sectioning and staining for relevant markers of cell proliferation and apoptosis.. Studies using colon cancer cells with gene disruptions suggested that the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway was essential for proliferation in 3-D culture. The dual PI3K-mTOR inhibitor NVP-BEZ235, currently in clinical trials, was found to inhibit phosphorylation of the mTOR target 4EBP1 in 3-D cultured cells. The ability of NVP-BEZ235 to inhibit tumour cell proliferation and to induce apoptosis was markedly more pronounced in 3-D cultures compared to monolayer cultures. It was subsequently found that NVP-BEZ235 was effective in inhibiting regrowth of 3-D cultured cells after treatment with two cytotoxic inhibitors of the ubiquitin-proteasome system (UPS), methyl-13-hydroxy-15-oxokaurenoate (MHOK) and bortezomib (Velcade®).. The dual PI3K-mTOR inhibitor NVP-BEZ235 was found to reduce cell proliferation and to induce apoptosis in 3-D cultured colon carcinoma cells, NVP-BEZ235 is a promising candidate for use in sequential treatment modalities together with cytotoxic drugs to reduce the cell mass of solid tumours.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma; Cell Proliferation; Cell Survival; Colonic Neoplasms; Flow Cytometry; Humans; Imidazoles; Immunohistochemistry; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Tumor Cells, Cultured

The mammalian target of rapamycin (mTOR) is frequently activated in colon cancers due to mutations in the phosphatidylinositol 3-kinase (PI3K) pathway. Targeting mTOR with allosteric inhibitors of mTOR such as rapamycin reduces colon cancer progression in several experimental models. Recently, a new class of mTOR inhibitors that act as ATP-competitive inhibitors of mTOR, has been developed. The effectiveness of these drugs in colon cancer cells has however not been fully characterized.. LS174T, SW480 and DLD-1 colon cancer cell lines were treated with PP242 an ATP-competitive inhibitor of mTOR, NVP-BEZ235, a dual PI3K/mTOR inhibitor or rapamycin. Tumor cell growth, proliferation and survival were assessed by MTS assay, 5-bromo-2'-deoxyuridine (BrDU) incorporation or by quantification of DNA fragmentation respectively. In vivo, the anticancer activity of mTOR inhibitors was evaluated on nude mice bearing colon cancer xenografts.. PP242 and NVP-BEZ235 reduced the growth, proliferation and survival of LS174T and DLD-1 colon cancer cells more efficiently than rapamycin. Similarly, PP242 and NVP-BEZ235 also decreased significantly the proliferation and survival of SW480 cells which were resistant to the effects of rapamycin. In vivo, PP242 and NVP-BEZ235 reduced the growth of xenografts generated from LS174T and SW480 cells. Finally, we also observed that the efficacy of ATP-competitive inhibitors of mTOR was enhanced by U0126, a MEK inhibitor.. Taken together, these results show that ATP-competitive inhibitors of mTOR are effective in blocking colon cancer cell growth in vitro and in vivo and thus represent a therapeutic option in colon cancer either alone or in combination with MEK inhibitors.

Topics: Animals; Antibiotics, Antineoplastic; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Female; Humans; Imidazoles; Immunohistochemistry; Indoles; Male; Mice; Protein Kinase Inhibitors; Purines; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays