p53 gene status and chemosensitivity in ovarian cancer.

Recent studies suggest that drug induced apoptosis relates to the sensitivity. p53 gene, which has a pivotal role in inducing apoptosis, frequently mutates in ovarian cancer. Therefore, p53 gene status and chemosensitivity in epithelial ovarian cancer is discussed. Nonresponders to chemotherapy had mutations of the p53 gene more frequently (83% for nonresponders vs. 16% for responders) in patients with epithelial ovarian cancer undergoing platinum-base chemotherapy. Apoptotic index was significantly greater in tumors with wild-type p53 gene than those without the gene. p53 gene transduction markedly enhanced the sensitivity to cisplatin (CDDP) and CDDP-induced apoptosis, but did not affect the sensitivity to paclitaxel (PTX) nor PTX-induced apoptosis in ovarian cancer cells without p53 gene. The combination treatment with a recombinant adenovirus carrying a wild-type p53 gene (AxCAp53) and CDDP significantly suppressed tumor growth of ovarian cancer cells with and without p53 gene, compared with a single treatment of either AxCAp53 or CDDP in ovarian cancer xenograft. Apoptotic index was significantly higher and proliferating cell nuclear antigen labeling index was relatively lower in an ovarian cancer xenograft without p53 gene receiving combination treatment, compared with a single treatment of either CDDP or AxCAp53, suggesting that the transduction of p53 gene induces apoptosis, but does not enhance the DNA repair system. A significant survival advantage was observed in the combination treatment compared with other treatments in carcinoma peritonitis models. In conclusion, p53 gene status contributes the sensitivity to CDDP in ovarian cancer. Additionally, combination treatment of p53 gene transduction and CDDP may be an effective therapeutic modality for ovarian cancer without wild-type p53 gene.     

Sensitization by glycerol for CDDP-therapy against human cultured cancer cells and tumors bearing mutated p53 gene

To clarify effective chemotherapeutic regimens against cancer, we examined the effects of glycerol on apoptosis induced by CDDP treatment using cultured human cancer cells (in vitro) and transplanted tumor in mice (in vivo). Human tongue cell carcinoma (SAS) cells transfected with mutated p53 gene (SAS/m p53) showed CDDP-resistance compared with the cells with neo control gene (SAS/ neo). When those cultured cells were pre-treated with glycerol, CDDP-induced apoptosis was enhanced by glycerol in SAS/m p53 cells but not in SAS/ neo cells.In tumor-transplanted mice, the glycerol treatment to tumors enhanced growth delay induced by CDDP in mp53 tumors transplanted with SAS/m p53 cells, but not in wtp53 tumors transplanted with SAS/ neo cells. When transplanted tumors were treated with CDDP alone, the cells positive for active caspase-3, 85 kDa PARP and apoptosis were observed by immunohistochemical staining in wtp53 tumors but not in mp53 tumors. When the tumors were treated with CDDP combined with glycerol, positive cells were observed not only in wtp53 tumors but also in mp53 tumors. These results showed that the CDDP-induced growth inhibition of the tumors is p53 -dependent and that the enhanced growth delay by glycerol may be due to the increased apoptosis. Glycerol might be available for cancer chemotherapy in patients with mp53 tumors.     

The Ganglioside GM3 Is Associated with Cisplatin-Induced Apoptosis in Human Colon Cancer Cells

Cisplatin (cis-diamminedichloroplatinum, CDDP) is a well-known chemotherapeutic agent for the treatment of several cancers. However, the precise mechanism underlying apoptosis of cancer cells induced by CDDP remains unclear. In this study, we show mechanistically that CDDP induces GM3-mediated apoptosis of HCT116 cells by inhibiting cell proliferation, and increasing DNA fragmentation and mitochondria-dependent apoptosis signals. CDDP induced apoptosis within cells through the generation of reactive oxygen species (ROS), regulated the ROS-mediated expression of Bax, Bcl-2, and p53, and induced the degradation of the poly (ADP-ribosyl) polymerase (PARP). We also checked expression levels of different gangliosides in HCT116 cells in the presence or absence of CDDP. Interestingly, among the gangliosides, CDDP augmented the expression of only GM3 synthase and its product GM3. Reduction of the GM3 synthase level through ectopic expression of GM3 small interfering RNA (siRNA) rescued HCT116 cells from CDDP-induced apoptosis. This was evidenced by inhibition of apoptotic signals by reducing ROS production through the regulation of 12-lipoxigenase activity. Furthermore, the apoptotic sensitivity to CDDP was remarkably increased in GM3 synthase-transfected HCT116 cells compared to that in controls. In addition, GM3 synthase-transfected cells treated with CDDP exhibited an increased accumulation of intracellular ROS. These results suggest the CDDP-induced oxidative apoptosis of HCT116 cells is mediated by GM3.     

p53 Is Required for Cisplatin-induced Processing of the Mitochondrial Fusion Protein L-Opa1 That Is Mediated by the Mitochondrial Metallopeptidase Oma1 in Gynecologic Cancers

Mitochondria are highly dynamic organelles, and mitochondrial fission is a crucial step of apoptosis. Although Oma1 is believed to be responsible for long form Opa1 (L-Opa1) processing during mitochondrial fragmentation, whether and how Oma1 is involved in L-Opa1 processing and participates in the regulation of chemoresistance is unknown. Chemosensitive and chemoresistant ovarian (OVCA) and cervical (CECA) cancer cells were treated with cisplatin (CDDP). Mitochondrial dynamics and protein contents were assessed by immunofluorescence and Western blot, respectively. The requirements of Oma1 and p53 for CDDP-induced L-Opa1 processing, mitochondrial fragmentation, and apoptosis were examined by siRNA or cDNA. CDDP induces L-Opa1 processing and mitochondrial fragmentation in chemosensitive but not in chemoresistant cells. CDDP induced Oma1 40-kDa form increases in OV2008 cells, not in C13* cells. Oma1 knockdown inhibited L-Opa1 processing, mitochondrial fragmentation, and apoptosis. Silencing p53 expression attenuated the effects of CDDP in Oma1 (40 kDa) increase, L-Opa1 processing, mitochondrial fragmentation, and apoptosis in chemosensitive OVCA cells, whereas reconstitution of p53 in p53 mutant or null chemoresistant OVCA cells induced Oma1 (40 kDa) increase, L-Opa1 processing, mitochondrial fragmentation, and apoptosis irrespective of the presence of CDDP. Prohibitin 1 (Phb1) dissociates from Opa1-Phb1 complex and binds phosphorylated p53 (serine 15) in response to CDDP in chemosensitive but not chemoresistant CECA cells. These findings demonstrate that (a) p53 and Oma1 mediate L-Opa1 processing, (b) mitochondrial fragmentation is involved in CDDP-induced apoptosis in OVCA and CECA cells, and (c) dysregulated mitochondrial dynamics may in part be involved in the pathophysiology of CDDP resistance.     

The α-mangostin prevention on cisplatin-induced apoptotic death in LLC-PK1 cells is associated to an inhibition of ROS production and p53 induction

Cisplatin (CDDP) is a widely useful chemotherapeutic agent for the treatment of tumors including lung, ovary and testis. Acute renal injury, however, is the main side effect observed after CDDP treatment. This side effect is related to the apoptotic death in proximal tubular cells in the kidney and p53 protein has a central role in this process. On the other hand, α-mangostin (α-M), a xanthone derived from the pericarp of mangosteen, exerts a renoprotective effect against cisplatin-induced renal damage in rats. The aim of this study was to evaluate whether α-M protects proximal tubule renal epithelial cells (LLC-PK1) from CDDP-induced apoptotic death. Cells were co-incubated with 5 μM α-M and 100 μM CDDP for 24 h. It was found that α-M attenuated the following alterations: the apoptotic cell death, the increase in reactive oxygen species (ROS), the glutathione depletion and the increase in p53 expression induced by CDDP. In conclusion, the preventive effect of α-M on CDDP-induced apoptotic death is associated to the inhibition of p53 expression and ROS generation.     

Overexpression of miR-22 reverses paclitaxel-induced chemoresistance through activation of PTEN signaling in p53-mutated colon cancer cells

Chemoresistance is a key cause of treatment failure in colon cancer. MiR-22 is a tumor-suppressing microRNA. To explore whether miR-22 is an important player in the development of chemoresistance in colon cancer, we overexpressed miR-22 and subsequently tested its role in cell proliferation, apoptosis, survival, and associated signaling in p53-mutated HT-29 and HCT-15 cells, and p53 wild-type HCT-116 cells. We further investigated the role of miR-22 on cytotoxicity of paclitaxel in both the p53-mutated and p53 wild-type colon cancer cells. Results showed that HT-29 and HCT-15 cells were resistant to paclitaxel-induced cytotoxicity, which normally inhibits cell proliferation and survival, and induces apoptosis. Conversely, HCT-116 was relatively sensitive to the cytotoxicity of paclitaxel. Overexpression of miR-22 significantly decreased cell proliferation and survival, and induced cell apoptosis in the p53-mutated colon cancer cells, but played no role in the p53 wild-type cells. Importantly, miR-22 overexpression enhanced the cytotoxic role of paclitaxel in p53-mutated HT-29 and HCT-15 cells, but not in p53 wild-type HCT-116 cell. We further demonstrated that the tumor-suppressive role of miR-22 in p53-mutated colon cancer cells was mediated by upregulating PTEN expression, which negatively regulated Akt phosphorylation at Ser(473) and MTDH expression, and subsequently increased Bax and active caspase-3 levels. Our study is the first to identify the tumor-suppressive role of miR-22 and its associated signaling in the p53-mutated colon cancer cells and highlighted the chemosensitive role of miR-22.     

beta-lapachone induces cell cycle arrest and apoptosis in human colon cancer cells

BACKGROUND: Human colon cancers have a high frequency of p53 mutations, and cancer cells expressing mutant p53 tend to be resistant to current chemo- and radiation therapy. It is thus important to find therapeutic agents that can inhibit colon cancer cells with altered p53 status. beta-Lapachone, a novel topoisomerase inhibitor, has been shown to induce cell death in human promyelocytic leukemia and prostate cancer cells through a p53-independent pathway. Here we examined the effects of beta-lapachone on human colon cancer cells. MATERIALS AND METHODS: Several human colon cancer cell lines, SW480, SW620, and DLD1, with mutant or defective p53, were used. The antiproliferative effects of beta-lapachone were assessed by colony formation assays, cell cycle analysis, and apoptosis analysis, including annexin V staining and DNA laddering analysis. The effects on cell cycle and apoptosis regulatory proteins were examined by immunoblotting. RESULTS: All three cell lines, SW480, SW620, and DLD1, were sensitive to beta-lapachone, with an IC(50) of 2 to 3 microM in colony formation assays, a finding similar to that previously reported for prostate cancer cells. However, these cells were arrested in different stages of S phase. At 24 hr post-treatment, beta-lapachone induced S-, late S/G2-, and early S-phase arrest in SW480, SW620, and DLD1 cells, respectively. The cell cycle alterations induced by beta-lapachone were congruous with changes in cell cycle regulatory proteins such as cyclin A, cyclin B1, cdc2, and cyclin D1. Moreover, beta-lapachone induced apoptosis, as demonstrated by annexin V staining, flow cytometric analysis of DNA content, and DNA laddering analysis. Furthermore, down-regulation of mutant p53 and induction of p27 in SW480 cells, and induction of pro-apoptotic protein Bax in DLD1 cells may be pertinent to the anti-proliferative and apoptotic effects of beta-lapachone on these cells. CONCLUSIONS: beta-Lapachone induced cell cycle arrest and apoptosis in human colon cancer cells through a p53-independent pathway. For human colon cancers, which often contain p53 mutations, beta-lapachone may prove to be a promising anticancer agent that can target cancer cells, especially those with mutant p53.     

The death domain kinase RIP has an important role in DNA damage-induced, p53-independent cell death

Tumor suppressor p53 plays a critical role in cellular responses, such as cell cycle arrest and apoptosis following DNA damage. DNA damage-induced cell death can be mediated by a p53-dependent or p53-independent pathway. Although p53-mediated apoptosis has been well documented, little is known about the signaling components of p53-independent cell death. Here we report that the death domain kinase, RIP (receptor-interacting protein), is important for DNA damage-induced, p53-independent cell death. DNA damage induces cell death in both wild-type and p53-/- mouse embryonic fibroblast cells. We found that RIP-/- mouse embryonic fibroblast cells, which have a mutant form of the p53 protein, are resistant to DNA damage-induced cell death. The reconstitution of RIP protein expression in RIP-/- cells restored the sensitivity of cells to DNA damage-induced cell death. We also found that RIP mediates this process through activating mitogen-activated protein kinase, JNK1. Furthermore, knocking down the expression of RIP blocked DNA damage-induced cell death in the human colon cancer cell line, p53 null HCT 116. Taken together, our study demonstrates that RIP is one of the critical components involved in mediating DNA damage-induced, p53-independent cell death.     

Brefeldin A Is a Potent Inducer of Apoptosis in Human Cancer Cells Independently of p53

Brefeldin A (BFA) is a natural product that affects the structure and function of the Golgi apparatus and is in development for cancer chemotherapy. We observed that a wide range of cancer cells could undergo DNA fragmentation associated with apoptosis after BFA treatment. This DNA fragmentation was induced within 15 h in HL60 leukemia cells and after 48 h in K562 leukemia and HT-29 colon carcinoma cells with BFA concentrations as low as 0.1 μM.The DNA fragmentation had the typical internucleosomal pattern in HL60 and HT-29 cells. Apoptotic cells were also detected by microscopy. BFA-induced apoptosis is p53-independent as HL60 and K562 cells are p53 null and HT-29 are p53 mutant cells. BFA could potentiate UCN-01 and staurosporine-induced DNA fragmentation in HL60 cells. Cyclin B1/Cdc2 kinase activity decreased after BFA treatment in HL60 cells, indicating that BFA-induced DNA fragmentation was independent of a cyclin B1/Cdc2 kinase upregulation pathway. Cycloheximide could not prevent BFA-induced DNA fragmentation in HL60 cells, suggesting that protein synthesis is not needed for HL60 cells to undergo apoptosis. On the contrary, cycloheximide blocked BFA-induced DNA fragmentation in HT-29 cells, indicating that apoptosis in HT-29 cells requires macromolecular synthesis. Cell-free system experiments suggested that cytosolic proteins play an important role in triggering DNA fragmentation during apoptosis induced by BFA. Our results show that transduction signaling pathways play central roles in apoptotic regulation.     

5-Aminolevulinic Acid Protects against Cisplatin-Induced Nephrotoxicity without Compromising the Anticancer Efficiency of Cisplatin in Rats In Vitro and In Vivo

Background/Aims

Nephrotoxicity is a frequent and major limitation in cisplatin (CDDP)-based chemotherapy. 5-Aminolevulinic acid (ALA) is widely distributed in animal cells, and it is a precursor of tetrapyrole compounds such as heme that is fundamentally important in aerobic energy metabolism. The aim of this study is to evaluate the protective role of ALA in CDDP-induced acute kidney injury (AKI).

Method

We used CDDP-induced AKI rat model and cultured renal tubular cells (NRK-52E). We divided four groups of rats: control, CDDP only, CDDP + ALA(post);(ALA 10 mg/kg + Fe in drinking water) after CDDP, CDDP + ALA(pre & post).

Result

CDDP increased Cr up to 6.5 mg/dl, BUN up to 230 mg/dl, and ALA significantly reduced these changes. ALA ameliorates CDDP-induced morphological renal damages, and reduced tubular apoptosis evaluated by TUNEL staining and cleaved caspase 3. Protein and mRNA levels of ATP5α, complex(COX) IV, UCP2, PGC-1α in renal tissue were significantly decreased by CDDP, and ALA ameliorates reduction of these enzymes. In contrast, Heme Oxigenase (HO)-1 level is induced by CDDP treatment, and ALA treatment further up-regulates HO-1 levels. In NRK-52E cells, the CDDP-induced reduction of protein and mRNA levels of mitochondrial enzymes was significantly recovered by ALA + Fe. CDDP-induced apoptosis were ameliorated by ALA + Fe treatment. Furthermore, we evaluated the size of transplantated bladder carcinoma to the rat skin, and ALA did not change the anti cancer effects of CDDP.

Conclusion

These data suggested that the protective role of ALA in cisplatin-induced AKI is via protection of mitochondrial viability and prevents tubular apoptosis. Also there are no significant effects of ALA on anticancer efficiency of CDDP in rats. Thus, ALA has the potential to prevent CDDP nephrotoxicity without compromising its anticancer efficacy.     

Differential Effects of Lovastatin on Cisplatin Responses in Normal Human Mesothelial Cells versus Cancer Cells: Implication for Therapy

The cancer killing efficacy of standard chemotherapeutic agents such as cisplatin (CDDP) is limited by their side effects to normal tissues. Therefore, research efforts optimizing the safety and efficacy of those agents are clinically relevant. We did screen for agents that specifically protect normal human mesothelial cells against CDDP without reducing the cancer cell killing efficacy. Lovastatin was identified from the screen. Lovastatin at a pharmacologically relevant concentration strongly arrested the proliferation of normal cells, whereas cancer cells were less affected. CDDP-induced DNA damage response was not activated and normal cells showed enhanced tolerance to CDDP when normal cells were treated with the combination of CDDP and lovastatin. We demonstrate that interfering with protein geranylgeranylation is involved in the lovastatin-mediated CDDP protective effect in normal cells. In contrast to normal cells, in cancer cells lovastatin did not change the CDDP-induced response, and cancer cells were not protected by lovastatin. Furthermore, lovastatin at the pharmacological relevant concentration per se induced DNA damage, oxidative stress and autophagy in cancer cells but not in normal mesothelial cells. Therefore, our data suggest that lovastatin has a potential to improve the therapeutic index of cisplatin-based therapy.     

Testis developmental related gene 1 regulates the chemosensitivity of seminoma TCam‐2 cells to cisplatin via autophagy

We previously identified testis developmental related gene 1 (TDRG1), a gene implicated in proliferation of TCam‐2 seminoma cells. Recent evidence has revealed that autophagy influences the chemosensitivity of cancer cells to chemotherapy. However, whether TDRG1 protein regulates autophagy in seminoma cells and influences their sensitivity to cis‐dichlorodiammine platinum (CDDP) remains unknown. In this study, we used TCam‐2 cells and male athymic BALB/c nude mice with xenografts of TCam‐2 cells to investigate autophagy, cell viability, apoptosis and the p110β/Rab5/Vps34 (PI3‐kinase Class III) pathway under the conditions of TDRG1 overexpression or knockdown and with or without CDDP treatment. We found that TDRG1 upregulation promoted autophagy in both TCam‐2 cells and seminoma xenografts via p110β/Rab5/Vps34 activation. Inhibition of autophagy reduced cell viability and promoted apoptosis during CDDP treatment of TCam‐2 cells. Similarly, TDRG1 knockdown inhibited autophagy, reduced cell viability and promoted apoptosis during CDDP treatment of TCam‐2 cells. TDRG1 knockdown inhibited tumour growth and promoted apoptosis in TCam‐2 cell xenografts, whereas TDRG1 overexpression had the opposite effect. According to these results, we propose that high expression of TDRG1 promotes autophagy through the p110β/Rab5/Vps34 pathway in TCam‐2 cells. TDRG1 overexpression promotes autophagy and leads to CDDP resistance, whereas TDRG1 knockdown inhibits autophagy and promotes chemosensitivity to CDDP both in vivo and in vitro. This study has uncovered a novel role of TDRG1 in reducing chemoresistance during CDDP treatment and provides potential therapeutic strategies for the treatment of human seminoma.     

MK2206 Enhances Cisplatin-Induced Cytotoxicity and Apoptosis in Testicular Cancer Through Akt Signaling Pathway Inhibition

OBJECTIVE: To improve conventional chemotherapeutic efficacy, it is significant to identify novel molecular markers for chemosensitivity as well as possible molecules accelerating cell-killing mechanisms. In this study, we attempted to elucidate how MK2206, an allosteric Akt inhibitor, enhances the cisplatin (CDDP)-induced cytotoxicity and apoptosis in testicular cancer. MATERIALS AND METHODS: We checked three testicular cancer cell lines for the expression of phospho(p)-Akt and its downstream molecules targets by Western blot. The potential antitumor effects were analyzed by MTT assay in vitro and by subcutaneous xenograft models in vivo. The cell invasion was analyzed by transwell invasion assay, and the activities of Akt signaling pathway and expression of apoptosis-related proteins were measured by Western blot. RESULTS: Our results indicated that there was overactivation of p-Akt and its downstream molecules in testicular cancer cell lines compared with normal testis epithelium cells. MK2206 (600 nM) inhibited cell invasion in TCAM-2 and P19 cell lines and significantly increased the susceptibility of testicular cancer to CDDP. Combined with CDDP, MK2206 potentiated CDDP-induced cytotoxicity and apoptosis, with repressed expression of p-Akt and its downstream targets. The subcutaneous xenograft models also showed that a combined CDDP/MK2206 therapy completely suppressed tumor growth without any side effects. CONCLUSION: These results suggested that the concomitant use of MK2206 could enhance the CDDP-induced cytotoxicity and apoptosis in testicular cancer with the suppressed expression of Akt pathway.     

Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G1/S checkpoint, ATM, and p53 signaling pathways in p53-wildtype cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G1/S checkpoint pathway in p53-wildtype lines and not in p53-mutant cells. These responses are coupled with G2/G1 checkpoint effectors p21CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G2 cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wildtype and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G2 arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wildtype and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell-cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.     

Transfection of mutant p53 gene depresses X-ray- or CDDP-induced apoptosis in a human squamous cell carcinoma of the head and neck

The present study examined whether X-ray- and CDDP-sensitivities depend on p53 gene status in human squamous cell carcinoma of the head and neck (SAS cells) showing dominant negative nature of mutant p53 protein. SAS cells were transfected with a vector carrying a mutant p53 gene (SAS/Trp248 cells) or neomycin resistant gene control vector (SAS/neo cells). Sensitivities of the transfected cells to X-ray or CDDP were measured with colony formation assay. The incidence of apoptosis by X-ray or CDDP was analyzed with Hoechst staining or DNA ladder formation assay. The activation of caspase-3 was estimated as an indicator of apoptosis by the detection of fragmentation of caspase-3 or poly (ADP ribose) polymerase (PARP) with Western blot. SAS/Trp248 cells showed X-ray- and CDDP-resistance due to the dominant negative nature of mutant p53, compared with SAS/neo cells. The incidence of DNA ladders and apoptotic bodies increased markedly in SAS/neo cells after X-ray irradiation or CDDP treatment, but increased only slightly in SAS/Trp248 cells. Fragmentation of caspase-3 and PARP was observed in SAS/neo cells, but almost no such fragmentation was observed in SAS/Trp248 cells after X-ray irradiation or CDDP treatment. The present results strongly suggest that the X-ray- and CDDP-sensitivities of human squamous cell carcinomas are p53-dependent, and that the sensitivities are tightly correlated with the induction of apoptosis through caspase-3 activation. The p53-dependent X-ray- or CDDP-sensitivity was supported by results from p53-null human lung cancer H1299 cells which were transfected with wild-type or mutant p53 gene.     

Sanguinarine causes DNA damage and p53-independent cell death in human colon cancer cell lines

The benzophenanthridine alkaloid sanguinarine has antimicrobial and possibly anticancer properties but it is not clear to what extent these activities involve DNA damage. Thus, we studied its ability to cause DNA single and double strand breaks, as well as increased levels of 8-oxodeoxyguanosine, in human colon cancer cells and found DNA damage consistent with oxidation. Since the tumor suppressor p53 is frequently involved in inducing apoptosis following DNA damage we investigated the effect of sanguinarine in wild type, p53-mutant and p53-null colon cancer cell lines. We found them to be equally sensitive to this plant compound, indicating that cell death is not mediated by p53 in this case. In addition, our observation that apoptosis induced by sanguinarine is initiated very rapidly raised the question whether there is enough time for cellular signaling in response to DNA damage. Moreover, the abundance of double strand breaks is not consistent with only oxidative damage to DNA. We conclude that the majority of DNA double strand breaks in sanguinarine-treated cells are likely the result, rather than the cause, of apoptotic cell death and that apoptosis induced by sanguinarine is independent of p53 and most likely independent of DNA damage.     

Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G₀/G₁ phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G₀/G₁ phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G₀/G₁ phase.     

Oxaliplatin down-regulates survivin by p38 MAP kinase and proteasome in human colon cancer cells

Oxaliplatin, a platinum derivative cancer drug, has been used for treating human colorectal cancers. Survivin has been proposed as a cancer target, which highly expressed in most cancer cells but not normal adult cells. In this study, we investigated the regulation of survivin expression by exposure to oxaliplatin in human colon cancer cells. Oxaliplatin (3–9 μM for 24 h) markedly induced cytotoxicity, proliferation inhibition and apoptosis in the human RKO colon cancer cells. The survivin protein expression of RKO cells is dramatically reduced by oxaliplatin; however, the survivin gene expression is slightly altered. The survivin blockage of oxaliplatin elevated caspase-3 activation and apoptosis in RKO cells. Over-expression of survivin proteins by transfection with a survivin-expressed vector resisted the oxaliplatin-induced cancer cell death. Meantime, oxaliplatin elicited the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB202190, a specific p38 MAP kinase inhibitor, restored the survivin protein level and attenuated oxaliplatin-induced cancer cell death. In addition, oxaliplatin increased the levels of phospho-p53 (Ser-15) and total p53 proteins. Inhibition of p53 expression by a specific p53 inhibitor pifithrin-α reduced the phosphorylated p38 MAP kinase and active caspase-3 proteins in the oxaliplatin-exposed RKO cells. In contrast, SB202190 did not alter the oxaliplatin-induced p53 protein level. Furthermore, treatment with a specific proteasome inhibitor MG132 restored survivin protein level in the oxaliplatin-treated colon cancer cells. Taken together, our results demonstrate for the first time that survivin is down-regulated by p38 MAP kinase and proteasome degradation pathway after treatment with oxaliplatin in the human colon cancer cells.