Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells

Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways.     

Selective inhibition of carbonic anhydrase-IX by sulphonamide derivatives induces pH and reactive oxygen species-mediated apoptosis in cervical cancer HeLa cells

Selective inhibition with sulphonamides of carbonic anhydrase (CA) IX reduces cell proliferation and induces apoptosis in human cancer cells. The effect on CA IX expression of seven previously synthesised sulphonamide inhibitors, with high affinity for CA IX, as well as their effect on the proliferation/apoptosis of cancer/normal cell lines was investigated. Two normal and three human cancer cell lines were used. Treatment resulted in dose- and time-dependent inhibition of the growth of various cancer cell lines. One compound showed remarkably high toxicity towards CA IX-positive HeLa cells. The mechanisms of apoptosis induction were determined with Annexin-V and AO/EB staining, cleaved caspases (caspase-3, caspase-8, caspase-9) and cleaved PARP activation, reactive oxygen species production (ROS), mitochondrial membrane potential (MMP), intracellular pH (pHi), extracellular pH (pHe), lactate level and cell cycle analysis. The autophagy induction mechanisms were also investigated. The modulation of apoptotic and autophagic genes (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin and LC3) was measured using real time PCR. The positive staining using γ-H2AX and AO/EB dye, showed increased cleaved caspase-3, caspase-8, caspase-9, increased ROS production, MMP and enhanced mRNA expression of apoptotic genes, suggesting that anticancer effects are also exerted through its apoptosis-inducing properties. Our results show that such sulphonamides might have the potential as new leads for detailed investigations against CA IX-positive cervical cancers.     

Daidzein Induced Apoptosis via Down-Regulation of Bcl-2/Bax and Triggering of the Mitochondrial Pathway in BGC-823 Cells

Daidzein belongs to the group of isoflavones, found in a wide variety of plant-derived foods, especially in soybeans and soy-based foods. In this study, the effect of daidzein on human gastric carcinoma cells (BGC-823) and its mechanism were investigated. MTT assay was applied in the detection of the inhibitory effects of daidzein on cell proliferation. Hoechst–propidium iodide staining and flow cytometry were used to examine the apoptosis as well as the mitochondrial transmembrane potential. Western blotting was performed to detect the expression of apoptosis-associated proteins: cleaved PARP, cleaved caspase-9, cleaved caspase-3, Bcl-2, and Bax. Daidzein significantly inhibited the growth and proliferation of human gastric carcinoma cells (BGC-823) in a concentration- and time-dependent manner. Furthermore, it was found that an insult of daidzein to BGC-823 cells caused them to die by disruption of mitochondrial transmembrane potential, demonstrated not only by staining dead cells for phosphatidylserine but also by the up-regulation (cleaved PARP, cleaved caspase-9, cleaved caspase-3, Bax) and down-regulation (Bcl-2) of proteins associated with apoptosis and survival; whereas, the pan-caspase inhibitor z-VAD-fmk could partially rescue cells against damage of daidzein. Taken together, the results of this study demonstrate that daidzein significantly induces apoptosis via a mitochondrial pathway. Specifically, daidzein induced a change in the Bax/Bcl-2 ratios and activation of caspases-3 and -9 and the cleavage of PARP. Therefore, daidzein has the potential for use as a therapeutic agent for the treatment of gastric carcinoma.     

LncRNA PVT1 predicts prognosis and regulates tumor growth in prostate cancer

Long non-coding RNA (lncRNA) plasmacytoma variant translocation 1(PVT1) was aberrantly expressed in various cancers and is associated with tumor prognosis. Here, we aim to investigate its function in prostate cancer. Small interfering RNA against PVT1 was transfected into prostate cancer cell lines and cell growth and apoptosis were analyzed. Our results showed that PVT1 was overexpressed in prostate cancer tissues and cells. Higher levels of PVT1 indicated poorer overall survival and disease-free survival. A significant association was found between PVT1 expression and tumor stage. Besides, PVT1 knockdown significantly inhibited prostate cancer growth in vivo and in vitro and promoted cell apoptosis. PVT1 knockdown also significantly upregulated the expression of cleaved caspase-3 and cleaved caspase-9, but downregulated the expression of c-Myc in prostate cancer cell lines. Our results suggest that PVT1 played an oncogenic role in prostate cancer and could be used as a potential biomarker for diagnosis of prostate cancer.     

Role of thymidine phosphorylase in Fas-induced apoptosis

Thymidine phosphorylase (TP) has chemotactic and angiogenic activity in vitro, and it promotes tumor growth and inhibits apoptosis in vivo. It plays a key role in the invasiveness and metastasis of TP-expressing solid tumors. KB/TP cells transfected with a TP cDNA have been shown to be resistant to hypoxia-induced apoptosis, suggesting that TP has effects on tumor growth and cell death independent of its effects on angiogenesis. However, the mechanisms of cell death inhibition by TP are unknown. In the present study, we demonstrate that caspase-8 is cleaved in control transfectant KB cells early on during Fas-induced apoptosis. Caspase-8 activation leads to the loss of mitochondrial membrane potential, followed by the release of cytochrome c, the activation of caspase-3, and apoptosis. In contrast, Fas-induced caspase-8 cleavage is inhibited in KB/TP cells, which lead to inhibition of the downstream apoptotic cascade and inhibition of apoptosis. These findings indicate that TP plays an important role in intracellular apoptotic signal transduction in the Fas-induced apoptotic pathway. Therefore, inhibition of TP may suppress the progression of TP-overexpressing solid tumors by inducing apoptosis.     

Antimetastatic activity of pinosylvin, a natural stilbenoid, is associated with the suppression of matrix metalloproteinases

Metastasis is a major cause of death in cancer patients. Our previous studies showed that pinosylvin, a naturally occurring trans-stilbenoid mainly found in Pinus species, exhibited a potential cancer chemopreventive activity and also inhibited the growth of various human cancer cell lines via the regulation of cell cycle progression. In this study, we further evaluated the potential antimetastatic activity of pinosylvin in in vitro and in vivo models. Pinosylvin suppressed the expression of matrix metalloproteinase (MMP)-2, MMP-9 and membrane type 1-MMP in cultured human fibrosarcoma HT1080 cells. We also found that pinosylvin inhibited the migration of HT1080 cells in colony dispersion and wound healing assay systems. In in vivo spontaneous pulmonary metastasis model employing intravenously injected CT26 mouse colon cancer cells in Balb/c mice, pinosylvin (10 mg/kg body weight, intraperitoneal administration) significantly inhibited the formation of tumor nodules and tumor weight in lung tissues. The analysis of tumor in lung tissues indicated that the antimetastatic effect of pinosylvin coincided with the down-regulation of MMP-9 and cyclooxygenase-2 expression, and phosphorylation of ERK1/2 and Akt. These data suggest that pinosylvin might be an effective inhibitor of tumor cell metastasis via modulation of MMPs.     

Nogo-B (Reticulon-4B) functions as a negative regulator of the apoptotic pathway through the interaction with c-FLIP in colorectal cancer cells

Nogo-B is a member of the Nogo/Reticulon-4 family and has been reported to be an inducer of apoptosis in certain types of cancer cells. However, the role of Nogo-B in human cancer remains less understood. Here, we demonstrated the functions of Nogo-B in colorectal cancer cells. In clinical colorectal cancer specimens, Nogo-B was obviously overexpressed, as determined by immunohistochemistry; and Western blot analysis showed its expression level to be significantly up-regulated. Furthermore, knockdown of Nogo-B in two colorectal cancer cell lines, SW480 and DLD-1, by transfection with si-RNA (siR) resulted in significantly reduced cell viability and a dramatic increase in apoptosis with insistent overexpression of cleaved caspase-8 and cleaved PARP. The transfection with Nogo-B plasmid cancelled that apoptosis induced by siRNogoB in SW480 cells. Besides, combinatory treatment with siR-Nogo-B/staurosporine (STS) or siR-Nogo-B/Fas ligand (FasL) synergistically reduced cell viability and increased the expression of apoptotic signaling proteins in colorectal cancer cells. These results strongly support our contention that Nogo-B most likely played an oncogenic role in colorectal cancer cells, mainly by negatively regulating the extrinsic apoptotic pathway in them. Finally, we revealed that suppression of Nogo-B caused down-regulation of c-FLIP, known as a major anti-apoptotic protein, and activation of caspase-8 in the death receptor pathway. Interaction between Nogo-B and c-FLIP was shown by immunoprecipitation and immunofluorescence studies.In conclusion, Nogo-B was shown to play an important negative role in apoptotic signaling through its interaction with c-FLIP in colorectal cancer cells, and may thus become a novel therapeutic target for colorectal cancer.     

Chetomin induces apoptosis in human triple-negative breast cancer cells by promoting calcium overload and mitochondrial dysfunction

Human triple-negative breast cancer (TNBC) is poorly diagnosed and unresponsive to conventional hormone therapy. Chetomin (CHET), a fungal metabolite synthesized by Chaetomium cochliodes, has been reported as a promising anticancer and antiangiogenic agent but the complete molecular mechanism of its anticancer potential remains to be elucidated. In our study, we explored the anti-neoplastic action of CHET on TNBC cells. Cytotoxicity studies were performed in human TNBC cells viz. MDA-MB-231 and MDA-MB-468 cells by Sulforhodamine B assay. It exhibited antiproliferative response and induced apoptosis in both the cell types. Cell cycle analysis revealed that it increases the sub G0/G1 phase cell population. Modulation of mitochondrial membrane potential, activation of caspase 3/7 and a remarkable increase in the expression of cleaved PARP and increased chromatin condensation was observed after CHET treatment in MDA-MB-231 and MDA-MB-468 cells. Additionally, an elevated level of intracellular Ca²⁺ played an important role in CHET mediated cell death response. Calcium overload in mitochondria led to release of cytochrome c which in turn triggered caspase-3 mediated cell death. Inhibition of calcium signalling using BAPTA-AM reduced apoptosis confirming the involvement of calcium signalling in CHET induced cell death. Chetomin also inhibited PI3K/mTOR cell survival pathway in human TNBC cells. The overall findings suggest that Chetomin inhibited the growth of human TNBC cells by caspase-dependent apoptosis and modulation of PI3K/mTOR signalling and could be used as a novel chemotherapeutic agent for the treatment of human TNBC in future.     

Suppression of Colorectal Cancer Liver Metastasis by Apolipoprotein(a) Kringle V in a Nude Mouse Model through the Induction of Apoptosis in Tumor-Associated Endothelial Cells

The formation of liver metastases in colorectal cancer patients is the primary cause of patient death. Current therapies directed at liver metastasis from colorectal cancer have had minimal impact on patient outcomes. Therefore, the development of alternative treatment strategies for liver metastasis is needed. In the present study, we demonstrated that recombinant human apolipoprotein(a) kringle V, also known as rhLK8, induced the apoptotic turnover of endothelial cells in vitro through the mitochondrial apoptosis pathway. The interaction of rhLK8 with glucose-regulated protein 78 (GRP78) may be involved in the induction of apoptosis because the inhibition of GRP78 by GRP78-specific antibodies or siRNA knockdown inhibited the rhLK8-mediated apoptosis of human umbilical vein endothelial cells in vitro. Next, to evaluate the effects of rhLK8 on angiogenesis and metastasis, an experimental model of liver metastasis was established by injecting a human colorectal cancer cell line, LS174T, into the spleens of BALB/c nude mice. The systemic administration of rhLK8 significantly suppressed liver metastasis from human colorectal cancer cells and improved host survival compared with controls. The combination of rhLK8 and 5-fluorouracil substantially increased these survival benefits compared with either therapy alone. Histological observation showed significant induction of apoptosis among tumor-associated endothelial cells in liver metastases from rhLK8-treated mice compared with control mice. Collectively, these results suggest that the combination of rhLK8 with conventional chemotherapy may be a promising approach for the treatment of patients with life-threatening colorectal cancer liver metastases.     

Quantitative imaging of apoptosis commitment in colorectal tumor cells

We have studied caspase-3 activation by combined DNA damage induction and EGFR kinase inhibition in order to identify potential EGFR-mediated survival signals conferring resistance to apoptosis in human colorectal tumor cells. The onset of apoptosis was microscopically imaged with a newly developed caspase-3 substrate sensor based on EGFP and tHcred1, enabling us to monitor caspase-3 activation in cells by fluorescence lifetime imaging microscopy or fluorescence correlation spectroscopy. Both optical approaches provide parameters quantitatively reporting the ratio between cleaved and uncleaved sensor, thereby facilitating the comparison of caspase-3 activation between different cells. Using these methods, we show that EGFR kinase inhibitors sensitize colorectal SW-480 tumor cells for 5-fluorouracil-induced apoptosis, indicating that EGFR-mediated survival signaling contributes to apoptosis resistance via its intrinsic kinase activity.     

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.     

Transcutaneous Carbon Dioxide Induces Mitochondrial Apoptosis and Suppresses Metastasis of Oral Squamous Cell Carcinoma In Vivo

Squamous cell carcinoma (SCC) is the main histological type of oral cancer. Its growth rate and incidence of metastasis to regional lymph nodes is influenced by various factors, including hypoxic conditions. We have previously reported that transcutaneous CO₂ induces mitochondrial apoptosis and decreases lung metastasis by reoxygenating sarcoma cells. However, previous studies have not determined the sequential mechanism by which transcutaneous CO₂ suppresses growth of epithelial tumors, including SCCs. Moreover, there is no report that transcutaneous CO₂ suppresses lymphogenous metastasis using human cell lines xenografts. In this study, we examined the effects of transcutaneous CO₂ on cancer apoptosis and lymphogenous metastasis using human SCC xenografts. Our results showed that transcutaneous CO₂ affects expressions of PGC-1α and TFAM and protein levels of cleavage products of caspase-3, caspase-9 and PARP, which relatives mitochondrial apoptosis. They also showed that transcutaneous CO₂ significantly inhibits SCC tumor growth and affects expressions of HIF-1α, VEGF, MMP-2 and MMP-9, which play essential roles in tumor angiogenesis, invasion and metastasis. In conclusion, transcutaneous CO₂ suppressed tumor growth, increased mitochondrial apoptosis and decreased the number of lymph node metastasis in human SCC by decreasing intra-tumoral hypoxia and suppressing metastatic potential with no observable effect in vivo. Our findings indicate that transcutaneous CO₂ could be a novel therapeutic tool for treating human SCC.     

Ganoderic acid T from Ganoderma lucidum mycelia induces mitochondria mediated apoptosis in lung cancer cells

Ganoderma lucidum is a well-known traditional Chinese medicinal herb containing many bioactive compounds. Ganoderic acid T (GA-T), which is a lanostane triterpenoid purified from methanol extract of G. lucidum mycelia, was found to exert cytotoxicity on various human carcinoma cell lines in a dose-dependent manner, while it was less toxic to normal human cell lines. Animal experiments in vivo also showed that GA-T suppressed the growth of human solid tumor in athymic mice. It markedly inhibited the proliferation of a highly metastatic lung cancer cell line (95-D) by apoptosis induction and cell cycle arrest at G(1) phase. Moreover, reduction of mitochondria membrane potential (Delta psi(m)) and release of cytochrome c were observed during the induced apoptosis. Our data further indicate that the expression of proteins p53 and Bax in 95-D cells was increased in a time-dependent manner, whereas the expression of Bcl-2 was not significantly changed; thus the ratio of Bcl-2/Bax was decreased. The results show that the apoptosis induction of GA-T was mediated by mitochondrial dysfunctions. Furthermore, stimulation of the activity of caspase-3 but not caspase-8 was observed during apoptosis. The experiments using inhibitors of caspases (Z-VAD-FMK, Z-DEVD-FMK and Z-IETD-FMK) confirmed that caspase-3 was involved in the apoptosis. All our findings demonstrate that GA-T induced apoptosis of metastatic lung tumor cells through intrinsic pathway related to mitochondrial dysfunction and p53 expression, and it may be a potentially useful chemotherapeutic agent.     

SIRT1 activation by SRT1720 attenuates bone cancer pain via preventing Drp1-mediated mitochondrial fission

Bone cancer pain (BCP) is the pain induced by primary bone cancer or tumor metastasis. Increasing evidence and our previous studies have shown that mammalian silent information regulator 2?homolog (SIRT1) is involved in periphery sensitization and central sensitization of BCP, and the underlying mechanism of SIRT1 in bone cancer pain may provide clues for pain treatment. Dynamin-related protein 1 (Drp1) is an essential regulator for mitochondrial fission. In this research, BCP model rats were established by injecting MRMT-1 rat mammary gland carcinoma cells into the left tibia of female Sprague-Dawley rats and validated by tibia radiographs, histological examination and mechanical pain test. As a result BCP rats exhibited bone destruction and sensitivity mechanical pain. BCP increased inflammatory cells infiltration and apoptosis, reduced SIRT1 protein expression and phosphorylation, and elevated Drp1 expression in spinal cord. An agonist of SIRT1 named SRT1720 intrathecal treatment in BCP rats increased SIRT1 phosphorylation, reduced the up-regulated Drp1 expression, and reversed pain behavior. SRT1720 also regulated Bcl-2/BAX and cleaved caspase-3 expressions, and inhibited mitochondrial apoptosis in spinal cord of BCP rats. For in vitro research, SRT1720 treatment decreased Drp1 expression in a dose-dependent manner, blocked CCCP-induced mitochondrial membrane potential change, consequently reduced apoptosis and promoted proliferation. These data suggest that SIRT1 activation by SRT1720 attenuated bone cancer pain via preventing Drp1-mediated mitochondrial fission. Our results provide new targets for therapeutics of bone cancer pain.     

Recombinant human PDCD5 sensitizes chondrosarcomas to cisplatin chemotherapy in vitro and in vivo

Clinical management of chondrosarcoma remains a challenging problem, largely due to the toxicity and resistance of this tumor to conventional chemotherapy. Programmed Cell Death 5 (PDCD5) is a protein that accelerates apoptosis in different cell types in response to various stimuli, and has been shown to be down-regulated in many cancer tissues. In this study, mRNA and protein levels of PDCD5 were found to be up-regulated in cisplatin-treated SW1353 chondrosarcoma cells compared with untreated cells. Recombinant human PDCD5 (rhPDCD5) was also shown to sensitize chondrosarcoma cells to cisplatin-based chemotherapy, with inhibition of cell growth and apoptosis detected both in vitro and in vivo. Increased expression of Bax and decreased expression of Bcl-2 were also observed, along with release of cytochrome c from mitochondria into the cytosol. Additionally, cleavage of caspase-9 and caspase-3, as well as the cleavage of poly (ADP-ribose) polymerase (PARP), were detected, suggesting that sensitization of chondrosarcoma cells involves the intrinsic mitochondrial apoptosis pathway. In vivo, the treatment of a xenograft model of chondrosarcoma with rhPDCD5 and cisplatin significantly inhibited tumor cell proliferation and induced apoptosis compared to treatment with cisplatin alone. Overall, these data provide a theoretical basis for the administration of rhPDCD5 and cisplatin for the treatment of patients with chondrosarcoma.     

Grape Seed Proanthocyanidins (GSPs) Inhibit the Growth of Cervical Cancer by Inducing Apoptosis Mediated by the Mitochondrial Pathway

Grape seed proanthocyanidins (GSPs), a biologically active component of grape seeds, have been reported to possess a wide array of pharmacological and biochemical properties. Recently, the inhibitory effects of GSPs on various cancers have been reported, but their effects on cervical cancer remain unclear. Here, we explored the effect of GSPs on cervical cancer using in vitro and in vivo models. In vitro, the treatment of HeLa and SiHa cells with GSPs resulted in a significant inhibition of cell viability. Further investigation indicated that GSPs led to the dose-dependent induction of apoptosis in cancer cells. The underlying mechanism was associated with increased expression of the pro-apoptotic protein Bak-1, decreased expression of the anti-apoptotic protein Bcl-2, the loss of mitochondrial membrane potential, and the activation of caspase-3, suggesting that GSPs induced cervical cancer cell apoptosis through the mitochondrial pathway. In addition, the administration of GSPs (0.1%, 0.2%, and 0.4%, w/v) as a supplement in drinking water significantly inhibited the tumor growth of HeLa and SiHa cells in athymic nude mice, and the number of apoptotic cells in those tumors was also increased significantly. Taken together, our studies demonstrated that GSPs could inhibit the growth of cervical cancer by inducing apoptosis through the mitochondrial pathway, which provides evidence indicating that GSPs may be a potential chemopreventive and/or chemotherapeutic agent for cervical cancer.