cinobufagin and Carcinoma--Hepatocellular

Cinobufagin, a cardiotonic steroid derived from toad venom extracts, exhibits significant anticancer properties by inhibiting Na[Formula: see text]/K[Formula: see text]-ATPase in cancer cells. It is frequently used in clinical settings to treat advanced-stage cancer patients, improving their quality of life and survival time. However, its long-term use can result in multidrug resistance to other chemotherapy drugs, and the exact mechanism underlying this effect remains unknown. Therefore, this study explores the molecular mechanism underlying the anticancer effects of cinobufagin in hepatocellular carcinomas (HCCs), specifically in HepG2 and Huh-7 cells. As determined using transcriptome analysis, cinobufagin-triggered protective autophagy suppressed cell apoptosis in liver cancer HepG2 and Huh-7 cells by inhibiting the phosphoinositide-3-Kinase (PI3K)-AKT serine/threonine kinase (AKT)-mammalian target of rapamycin (mTOR) pathway. Cinobufagin-inhibited cell proliferation, induced apoptosis, and generated cell autophagy by upregulating the expression of MAP1 light chain 3 protein II, Beclin1, and autophagy-related protein 12-5. In addition, the autophagy inhibitor MRT68921 improved the antiproliferative and proapoptotic effects of cinobufagin in the studied cell lines. Overall, this study suggests that combining cinobufagin with an autophagy inhibitor can effectively treat HCC, providing a potential strategy for cancer therapy.

Topics: Amphibian Venoms; Apoptosis; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-akt; Quality of Life

Anti-tumor candidate drugs from natural products have gained increasing attention. Cinobufagin is a natural product isolated from the traditional chinese medicine Chansu. Herein, we find that cinobufagin inhibits the proliferation and colony-forming ability of human hepatoma HepG2 and SK-HEP-1 cells. Furthermore, cinobufagin induces G2-phase cell cycle arrest and DNA damage in cancer cells. Thymidylate synthase (TYMS), the major target of chemotherapeutic drugs 5-FU or other fluoropyrimidines, which catalyzes the conversion of dUMP to dTMP and provides the sole de novo source of thymidylate for DNA synthesis. We demonstrate that cinobufagin suppresses TYMS expression via proteasome-dependent degradation in human hepatoma cells, moreover, depletion of TYMS restrains the proliferation and colony formation of tumor cells, and the results of western blotting and immunofluorescence assay indicate DNA damage is induced in tumor cells transfected with TYMS-targeting siRNA (siTYMS), additionally, knockdown of TYMS enhances the inhibitory effect of cinobufagin on the proliferative potential of HepG2 and SK-HEP-1 cells. It is worth noting that cinobufagin in combination with 5-FU exhibits antagonism or synergism combined effects on the proliferation of human hepatoma cells, indicating that Chansu-related preparations such as cinobufacini injection and Huachansu capsules applied to clinical practice should be used with caution in combination with 5-FU for the treatment of liver cancer. Collectively, cinobufagin exerts good anti-hepatoma activity through inhibition of growth and induction of DNA damage by promoting the degradation of TYMS. Our results provide evidence that cinobufagin might be a potential agent for the treatment of cancers such as hepatocellular carcinoma. It can also promote the scientific development of Chansu, and has great significance for enriching the application of TCM in the development of new anti-tumor drugs.

Topics: Amphibian Venoms; Antineoplastic Agents; Bufanolides; Carcinoma, Hepatocellular; Cell Proliferation; DNA Damage; Fluorouracil; Humans; Liver Neoplasms; Proteasome Endopeptidase Complex; Thymidylate Synthase

Cinobufagin is one of the pharmaceutically active ingredients in the parotoid glands of the Chinese toad Bufo bufo gargarizans Cantor. This study was conducted to investigate the effect of cinobufagin on viability, migration, and apoptosis of hepatocellular carcinoma (HCC) cells and its mechanisms. Human HCC cells (HepG2) were treated with cinobufagin and assessed for viability, apoptosis, and migration using CCK-8 assay, flow cytometry, and wound healing assay. The expression of genes related to the p53, AKT, and ERK pathways was detected using RT-PCR and Western blot analysis. Cell viability assays showed that cinobufagin reduced the viability of HepG2 cells in a concentration- and time-dependent manner. Significantly increased apoptosis was detected in cinobufagin-treated cells as compared with non-treated cells. The migration ability of HepG2 cells was significantly reduced after they were exposed to cinobufagin as compared with control. RT-PCR and Western blot analyses showed that the expression levels of p53, caspase-3, and Bax were significantly upregulated, and the expression levels of AKT and ERK were significantly downregulated after cinobufagin treatment. Our data demonstrated that cinobufagin reduces the viability and induces apoptosis of HepG2 cells. The cytotoxicity is likely achieved by upregulating the p53 pathway and downregulating the Akt and ERK pathways.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Signaling System; Proto-Oncogene Proteins c-akt; Tumor Suppressor Protein p53

Hepatocellular carcinoma (HCC) is one of the deadliest cancers with high mortality and poor prognosis, and the investigation on new approaches and effective drugs for HCC therapy is of great significance. In our study, we demonstrate that treatment with cinobufagin, a natural compound isolated from traditional chinese medicine Chansu, reduces proliferation and the colony formation capacity of the human hepatoma cells in vitro, in addition, cinobufagin induces mitotic arrest in human hepatoma cells. The results of a network pharmacology-based analysis show that EGFR, MAPK1, PTK2, CDK2, MAPK3, ESR1, CDK1, PRKCA, AR, and CSNK2A1 are the key targets involved in the anti-tumor activities of cinobufagin, additionally, several signaling pathways such as proteoglycans in cancer, pathways in cancer, HIF-1 signaling pathway, VEGF signaling pathway, ErbB signaling pathway, and PI3K-AKT signaling pathway are identified as the potential pathways involved in the inhibitory effects of cinobufagin against HCC. Furthermore, at the molecular level, we find that cinobufagin decreases EGFR expression and CDK2 activity in human hepatoma cells. Inhibition of EGFR or CDK2 expression could not only suppress the growth of tumor cells but also enhance the inhibitory effects of cinobufagin on the proliferative potential of human hepatoma cells. We also demonstrate that EGFR positively regulates CDK2 expression. Furthermore, EGFR inhibitor gefitinib or CDK2 inhibitor CVT-313 synergistically enhances anticancer effects of cinobufagin in human hepatoma cells. Taken together, these findings indicate that cinobufagin may exert antitumor effects by suppressing EGFR-CDK2 signaling, and our study suggests that cinobufagin may be a novel, promising anticancer agent for the treatment of HCC.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Bufanolides; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin-Dependent Kinase 2; Down-Regulation; Drug Synergism; ErbB Receptors; Gefitinib; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Hep G2 Cells; Humans; Liver Neoplasms; M Phase Cell Cycle Checkpoints; Network Pharmacology; Protein Interaction Maps; Protein Kinase Inhibitors; Purines; Signal Transduction

Hepatoma is a common malignancy in the world with high morbidity and mortality. The treatment of hepatoma is limited by its poor response to many chemotherapeutic agents. Although paclitaxel (PTX) is widely used in clinical chemotherapy, the low sensitivity to hepatoma restricts its application. Combination therapy is a promising approach to resolve this dilemma.. To evaluate the interaction between paclitaxel, bufalin (BFL) and cinobufagin (CBF), and explore the optimum combination efficiently.. HepG2 cells were treated with PTX, BFL and CBF individually or in combination. Their interactions were evaluated by two classical models (Chou-Talalay model and Bliss independence). Response surface methodology (RSM) was used to explore the optimum combination. Furthermore, the optimum drug combination was verified by the morphological experiment.. Synergistic effects were observed when cells were exposed to binary mixtures of PTX+CBF and BFL+CBF. Although the interaction of PTX and BFL was summative, a strong synergistic effect was observed when cells were exposed to ternary mixtures of PTX+BFL+CBF. The interaction results of RSM were consistent with classical models, but more efficient. Moreover, the optimum combination dose was given by RSM without the combinatorial explosion of exhaustive testing.. The combination of BFL and CBF synergistically enhanced the potency of PTX against HepG2 cells. RSM could give an accurate evaluation for drug interactions and efficient prediction of optimum combination.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Bufanolides; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Drug Screening Assays, Antitumor; Hep G2 Cells; Humans; Liver Neoplasms; Molecular Conformation; Paclitaxel; Surface Properties; Tumor Cells, Cultured

The Na+/K+‑ATPase inhibitor cinobufagin exhibits numerous anticancer effects on hepatocellular carcinoma (HCC) cells expressing wild‑type p53 via inhibition of aurora kinase A (AURKA) and activation of p53 signaling. However, the effects of cinobufagin on HCC cells expressing mutant p53 remain unclear. In the present study, the anticancer effects of cinobufagin were investigated on HCC Huh‑7 cells with mutant p53, and the effects of AURKA overexpression or inhibition on the anticancer effects of cinobufagin were analyzed. Viability, cell cycle progression and apoptosis of cells were determined using an MTT assay, flow cytometry and Hoechst 33342 staining, respectively. The expression levels of p53 and p73 signaling‑associated proteins were investigated via western blot analysis. The results demonstrated that the expression levels of AURKA, B‑cell lymphoma 2 (Bcl‑2), cyclin‑dependent kinase 1, cyclin B1, proliferating cell nuclear antigen and heterogeneous nuclear ribonucleoprotein K, as well as the phosphorylation of p53 and mouse double minute 2 homolog, were significantly decreased in Huh‑7 cells treated with 5 µmol/l cinobufagin for 24 h. Conversely, the expression levels of Bcl‑2‑associated X protein, p21, p53 upregulated modulator of apoptosis and phorbol‑12‑myristate‑13‑acetate‑induced protein 1, were significantly increased by cinobufagin treatment. Overexpression or inhibition of AURKA suppressed or promoted the anticancer effects of cinobufagin on Huh‑7 cells, respectively. These results indicated that cinobufagin may induce anticancer effects on Huh‑7 cells via the inhibition of AURKA and p53 signaling, and via the activation of p73 signaling, in an AURKA‑dependent manner.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Aurora Kinase A; Bufanolides; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Humans; Liver Neoplasms; Signal Transduction; Tumor Protein p73; Tumor Suppressor Protein p53

To investigate the mechanism of cinobufagin-reduced cancer pain in mouse cancer pain model and in vitro cell co-culture system.. Female Kunming mice were randomly divided into 4 groups. One group of animals was set as normal control without any treatment. Other three groups of animals received H22 hepatoma cell inoculation in right hind paw. At day 9 after inoculation, mice in other three groups were injected intraperitoneally once a day for 8 days with the solvent, morphine or cinobufagin, respectively. The pain behavior was recorded daily. On the last day, all mice were sacrificed and xenograft tissues homogenate and plasma levels of β-endorphin (β-END), corticotropin-releasing factor (CRF) and interleukin-1β (IL-1β) were assessed by ELISA assay. Immunohistochemistry was performed to determine the expression of β-END, pro-opiomelanocortin (POMC) and the μ-opioid receptor (μ-OR) in the xenograft tissues. Immunofluorescence was used to localize lymphocytes with expression of CD3+, CD4+ and CD8+ in xenograft tumors and adjacent tissues. Mice splenic lymphocytes and H22 hepatoma carcinoma ascites cells were prepared for co-culture. β-END and CRF were detected in co-culture supernatants. The MTT assay and cytometry were used to assess cell proliferation. RT-PCR was conducted to determine the gene expression of POMC and Cathepsin L (CTSL). Chemotaxis was examined using a transwell-based migration assay.. Compared to the model group, the thermal and mechanical pain thresholds were increased in mice after cinobufagin treatment. The expression of β-END and CRF in the plasma and tumor tissues of cinobufagin group were much higher than that of the model group mice, but the expression of IL-1β in the plasma and tumor tissues was much lower than that in the model group mice. Meanwhile, the expression of β-END, POMC and μ-OR proteins was significantly increased in the xenograft tissues from cinobufagin group. Lymphocyte population of CD3+, CD4+, CD8+ were also elevated in xenograft tumors and adjacent tissues. In the cell co-culture assays, the content of β-END in the supernatant was significantly increased by cinobufagin in a dose-dependent manner. Cinobufagin also largely increased the proliferation of immune cells and inhibited H22 hepatoma carcinoma cell proliferation in single or co-culture cell assays. Gene expression of POMC and CTSL in cinobufagin group was significantly up-regulated comparing to the control group. Finally, cinobufagin addition enhanced the migration of immune cells in transwell assay.. Cinobufagin-induced local analgesic effect might be associated with increased activity of POMC/β-END/μ-OR pathway released from invaded CD3/4/8 lymphocytes in cancer tissues.

Topics: Analgesics; Animals; beta-Endorphin; Bufanolides; Carcinoma, Hepatocellular; Cell Line, Tumor; Coculture Techniques; Corticotropin-Releasing Hormone; Disease Models, Animal; Drugs, Chinese Herbal; Enzyme-Linked Immunosorbent Assay; Female; Immunohistochemistry; Interleukin-1beta; Liver Neoplasms; Lymphocytes, Tumor-Infiltrating; Mice; Neoplasms, Experimental; Pain; Pain Threshold; Polymerase Chain Reaction; Random Allocation

Cinobufacini has been traditionally used in China for the treatment of tumor since hundreds years ago. For recent years, its modern preparation,cinobifucini injection has also obtained satisfactory therapeutic functions for cancer.. High performance liquid chromatography (HPLC) analysis was applied to determine the content of cinobufagin, resibufogenin and bufothionine in cinobufacin extract liquid and injection; MTT assay and flow cytometric analysis were also respectively used to study the effect of cinobufacini extract liquid, injection and three chemical structures on cells and cell cycles.. HPLC results demonstrated that in cinobufacini extract liquid three ingredients (cinobufagin, resibufogenin and bufothionine) were all monitored while in cinofacini injection only bufothinone was detected; MTT assays showed bufothionine could obviously inhibit the proliferation of human hepatocellular carcinoma cell lines such as SMMC-7721 and BEL-7402 in a dose- and time-dependent manner as well as cinobufagin and resibufogenin; further flow cytometric analysis indicated obvious increases in G2/M phase and decrease in G0/G1 phase when SMMC-7721 cell line exposure to bufothionine (480 μg/ml).. These results suggested bufothionine could be involved in treatment of human cancer for cinobufacini injection and the mechanism might be relative to induce G2/M phase cell cycle arrest.

Topics: Amphibian Venoms; Antineoplastic Agents; Bufanolides; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Flow Cytometry; G2 Phase Cell Cycle Checkpoints; Humans; Indole Alkaloids; Injections; Liver Neoplasms; Medicine, Chinese Traditional; Quinolinium Compounds; Time Factors

Recent studies revealed the potential of Na(+)/K(+)-ATPase as a target for anticancer therapy and showed additional modes of action of cardiotonic steroids (CSs), a diverse family of naturally derived compounds, as inhibitors of Na(+)/K(+)-ATPase. The results from epidemiological studies showed significantly lower mortality rates in cancer patients receiving CSs, which sparked interest in the anticancer properties of these drugs. The present study was designed to investigate the anticancer effect of CSs (ouabain or cinobufagin) and to elucidate the molecular mechanisms of CS activity in hepatoma cell lines (HepG2 and SMMC-7721). Ouabain and cinobufagin significantly inhibited cell proliferation by attenuating the phosphorylation of extracellular regulated kinase (ERK) and down-regulating the expression of C-myc. These CSs also induced cell apoptosis by increasing the concentration of intracellular free calcium ([Ca(2+)](i)) and induced S phase cell cycle arrest by down-regulating the expression of Cyclin A, cyclin dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) as well as up-regulating the expression of cyclin dependent kinase inhibitor 1A (p21(CIP1)). Overexpression of ERK reversed the antiproliferation effect of ouabain or cinobufagin in HepG2 and SMMC-7721 cells. Currently, the first generation of CS-based anticancer drugs (UNBS1450 and Anvirzel) are in Phase I clinical trials. These data clearly support their potential use as cancer therapies.

Topics: Apoptosis; Blotting, Western; Bufanolides; Calcium; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin A; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Extracellular Signal-Regulated MAP Kinases; Hep G2 Cells; Humans; Microscopy, Confocal; Ouabain; Phosphorylation; Proliferating Cell Nuclear Antigen; Reverse Transcriptase Polymerase Chain Reaction

Bufadienolides bufalin and cinobufagin are cardiotonic steroids isolated from the skin and parotid venom glands of the toad Bufo bufo gargarizans Cantor. They have been shown to induce a wide spectrum of cancer cell apoptosis. However, the detailed molecular mechanisms of inducing apoptosis in hepatocellular carcinoma (HCC) are still unclear. In the present study, the apoptosis-inducing effect of bufalin and cinobufagin on HCC cell line HepG(2) was investigated. We found bufalin and cinobufagin induced marked changes in apoptotic morphology and significantly increased the proportion of apoptotic cells. This apoptotic induction was associated with an increase in Fas, Bax and Bid expression, a decrease in Bcl-2 expression, disruption of the mitochondrial membrane potential, release of cytochrome c, activation of caspase-3, -8, -9 and -10, and the cleavage of poly(ADP-ribose)polymerase (PARP), which indicated that bufalin and cinobufagin induced apoptosis through both Fas- and mitochondria-mediated pathways. In addition, caspase activation during bufalin- and cinobufagin-induced apoptosis was further confirmed by caspase-3 inhibitor Z-DEVD-FMK, caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHD-FMK and caspase-10 inhibitor Z-AEVD-FMK. The results showed that bufalin- and cinobufagin-induced apoptosis was blocked by these inhibitors and particularly by caspase-10 inhibitor. Taken together, bufalin and cinobufagin induce apoptosis of HepG(2) cells via both Fas- and mitochondria-mediated pathways, and a Fas-mediated caspase-10-dependent pathway might play a crucial role.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Bufanolides; Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Liver Neoplasms; Mitochondria; Signal Transduction

Cinobufagin (1) was one of important cardenolidal steroids and major components of Chan'Su, a famous traditional Chinese medicine. Bioconversion of cinobufagin by the fungi of Mucor spinosus and Aspergillus niger were investigated. Nine bioconversion products were obtained from M. spinosus and seven products from A. niger. Their structures were elucidated by high-resolution fast atom bombardment mass spectroscopy (HR-FAB-MS), extensive NMR techniques, including (1)H NMR, (13)C NMR, DEPT, (1)H-(1)H correlation spectroscopy (COSY), two-dimensional nuclear Overhauser effect correlation spectroscopy (NOESY), heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond coherence (HMBC). The in vitro cytotoxic activities against human hepatoma cells (HepG2, SMMC-7221 and BEL-7402) and human leukemia cells (K562, HL-60 and HEL) of all bioconversion products were determined by the MTT method, and their structure-activity relationships (SAR) were discussed.

Topics: Antineoplastic Agents; Aspergillus niger; Biotransformation; Bufanolides; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Humans; Leukemia; Molecular Conformation; Mucor; Stereoisomerism; Structure-Activity Relationship; Tumor Cells, Cultured