Topoisomerase I inhibitor (camptothecin)-induced apoptosis in human gastric cancer cells and the role of wild-type p53 in the enhancement of its cytotoxicity

Camptothecin (CPT), a human topoisomerase I inhibitor, blocks DNA replication in human cancer cells. It represents a promising new class of chemotherapeutic agents with broad anti-tumor activity. However, its effect on gastric cancer cells remains unknown. We examined cell growth, apoptosis and cell cycle phase distribution in gastric cancer cells by exposing these cells to CPT for up to 72 h. Cell viability was determined by the Trypan blue exclusion assay. Cell cycle phase distribution and apoptosis were measured using flow cytometry, fluorescence microscopy and DNA ladder assay. Exposure of exponentially growing gastric AGS cancer cells to CPT induced time-dependent apoptosis and growth inhibition. Serum starvation-synchronized AGS cells (about 60% cells in G0/G1 phase) showed similar cellular responses. Analysis of cell cycle phase distribution of AGS cells treated with CPT for up to 72 h showed no obvious differences compared to untreated control cells. Although the induction of apoptosis was noticed in gastric cancer cell lines both with and without p53, cells lacking p53 showed less apoptosis compared to those cell lines possessing p53. Our data show that CPT is capable of inducing gastric cancer cell growth inhibition and apoptosis. Wild-type p53 may enhance the cytotoxicity of CPT against gastric carcinoma.     

Ras mutation, irrespective of cell type and p53 status, determines a cell's destiny to undergo apoptosis by okadaic acid, an inhibitor of protein phosphatase 1 and 2A.

Okadaic acid (OA), a toxin from the black sponge Halicondria okadai, is a specific inhibitor of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A). OA is a tumor promoter but also induces apoptosis in some tumor cell lines. In this study, we determined whether ras mutation and/or p53 status are characteristics associated with the cell's sensitivity to the induction of apoptosis by OA. Several cell lines that differed in ras and p53 mutations were treated with OA (10-100 nM). At 24 to 48 h after treatment, the percentage of cells undergoing apoptosis was quantitated. The cell lines with mutations in either H-ras (human bladder carcinoma cell line T24 and mouse keratinocyte cell line 308), or K-ras (human colon carcinoma cell lines DLD-1 and HCT116; human prostate cancer cell lines LNCaP and PC-3; human lung cancer cell lines Calu-6 and SKLU-1; and human pancreatic cancer cell line MIAPaCa2) were more sensitive to OA-induced apoptosis (3- to 10-fold) than the cell lines that lacked the ras mutation (mouse epidermal cell lines C50 and JB6; murine fibroblast cell line NIH3T3; human colon cancer cell line HT29; human kidney epithelial cell line Hs715.K; and human pancreatic cancer cell line Bx-PC3). Similarly, using isogenic cell lines we found that overexpression of mutated H-ras in NIH3T3 and in SV40 immortalized human uroepithelial cells (SVHUC) enhanced their sensitivity to undergo apoptosis in response to OA treatment. The T24, DLD-1, SKLU-1, Calu-6, and MIAPaCa2 cell lines express mutated p53. The SVHUC as well as their ras-transfected counterparts have inactive p53 due to complex formation between large "T" antigen and p53. Taken together, these results imply that OA-induced apoptosis may involve a p53-independent pathway. The transfectants (NIH3T3-ras and SVHUC-ras), which express mutated H-ras, have up-regulated PP2A activity. OA treatment inhibited in vivo the levels of PP1 and PP2A activity, and induced apoptosis in SVHUC-ras and other cell lines. We conclude that OA-induced cell death pathway in ras-activated cell lines may involve a cross talk between PP1 and PP2A and ras signaling pathways. In light of the present results, the current theory that OA promotes mouse skin tumor formation by selective expansion of initiated cells that harbor ras mutations needs reevaluation.     

Cell cycle arrest and proapoptotic effects of the anticancer cyclodepsipeptide serratamolide (AT514) are independent of p53 status in breast cancer cells

In a search for new anticancer agents, we have identified serratamolide (AT514), a cyclodepsipeptide from Serratia marcescens 2170 that induces cell cycle arrest and apoptosis in various cancer cell lines. A cell viability assay showed that the concentrations that cause 50% inhibition (IC50) in human cancer cell lines range from 5.6 to 11.5 microM depending on the cell line. Flow cytometry analysis revealed that AT514 caused cell cycle arrest in G0/G1 or cell death, depending on the cell type and the length of time for which the cells were exposed to the drug. Subsequent studies revealed that AT514-induced cell death is caused by apoptosis, as indicated by caspases activation (8, 9, 2 and 3) and cleavage of poly (ADP-ribose) polymerase (PARP), release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, and the appearance of apoptotic bodies and DNA laddering. Alterations in protein levels of Bcl-2 family members might be involved in the mitochondrial disruption observed. AT514 induced p53 accumulation in wild-type p53 cells but cell death was observed in both deficient and wild-type p53 cells. Our results indicate that AT514 induces cell cycle arrest and apoptosis in breast cancer cells irrespectively of p53 status, suggesting that it might represent a potential new chemotherapeutic agent.     

p53 dependent and independent sensitivity to oxaliplatin of colon cancer cells

Oxaliplatin is an efficient chemotherapeutic agent used for the treatment of metastatic human colon cancer, but cancer cells are frequently resistant. The aim of this study was to analyse the underlying mechanisms in a panel of 10 human colorectal cancer cell lines submitted to a short (2h) oxaliplatin treatment period, accordingly to the usual therapeutic procedure in humans. Sensitivity to oxaliplatin was a characteristic of p53 wild-type colon cancer cells. In contrast, all p53-mutated cell lines had a high IC50 to oxaliplatin, with the exception of the V9P cell line. Exposure to oxaliplatin resulted in G0/G1 arrest in p53 wild-type cell lines, and in S phase in p53-mutated cell lines. In our treatment conditions, no DNA accumulation in sub G0/G1 phase, no caspase-3 activation nor PARP cleavage were detected after oxaliplatin treatment, except for the V9P cell line. The major role of the p53-p21 pathway in oxaliplatin sensitivity was confirmed in the p53 wild-type HCT116 cell line, using siRNA duplex, and knockdown of the TAp73 protein also enhanced resistance to oxaliplatin in this cell line. Surprisingly, siRNA duplex invalidation revealed a residual effect of the mutant p53 protein in p53-mutated cell lines. Persistent sensitivity to oxaliplatin of the p53-mutated V9P cell line was associated with oxalipatin-induced apoptosis but TAp73 was not the responsible alternative pathway.     

Octahedral Pt(IV) complex K101 induces apoptosis via ERK1/2 activation and the p53 pathway in human colon cancer cells

Recently, the synthesized octahedral Pt(IV) compound trans,cis-Pt(acetato)2Cl2(1,4-butanediamine), K101, showed potent anti-tumor activity in vitro and in vivo. For the further investigation of K101-induced anti-cancer activity, we tested cytotoxicity against various cancer cell lines and performed the histoculture drug response assay (HDRA) against human colorectal tumor tissues in vitro. We investigated the signaling pathway of K101-induced apoptosis via expression of p53 and ERK1/2 in the human colon cell line HCT116. The cytotoxicity and the three-dimensional HDRA of K101 were evaluated using the MTT assay. To study the K101-induced apoptosis pathway, we performed FACS analysis and immunoblotting of p53, p21, Bax, Fas and ERK1/2 in HCT116 cells treated with or without K101. The cytotoxic IC50 values of K101 ranged from 1.15 to 2.38 micromol/l, compared to cisplatin ranging from 2.13 to 13.1 micromol/l. Among several cancer cell lines, K101 showed greater potency than cisplatin in colon cancer cell lines. In the HDRA, K101 showed 80.0-91.4% efficacy rates compared with 48.6% for cisplatin against colorectal cancer patient tissues. In the signaling pathway, the expression of p53 and phospho-ERK1/2 was increased in a time-dependent manner by treatment with K101 in the HCT116 cells. When K101 was treated with MEK inhibitor U0126, the cell death rate was increased. The octahedral Pt(IV) complex K101 could be an attractive candidate as a chemotherapeutic agent against colon cancer. ERK1/2 activation and the p53 pathway may play significant functions in mediating K101-induced apoptosis in human colon cancer cells.     

Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53

Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X(L), Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.     

Efficient induction of apoptosis by ONYX-015 adenovirus in human colon cancer cell lines regardless of p53 status

The ONYX-015 virus is a mutated adenovirus that in theory selectively replicates and induces cytolysis in tumor cells lacking functional p53. The present study investigated whether ONYX-015 viral infection alone or in combination with conventional chemotherapeutic agents could significantly increase apoptosis in human colon cancer cell lines, regardless of p53 status, compared to untreated cells. A pair of colon cancer cell lines that differ only in their p53 status (RKO with wild-type p53 and RKOp53 with deficient p53) was tested. Two chemotherapeutic agents, 5-fluorouracil (5-FU) and CPT-11, were tested in combination with ONYX-015. Final concentrations of these agents corresponded to peak plasma levels achievable in patients. ONYX-015 concentration was 10 p.f.u./cell. In RKO and RKOp53 cell lines, ONYX-015 viral infection alone or in combination with 5-FU or CPT-11 induced a significant increase in apoptosis compared to chemotherapeutic agents alone, regardless of p53 status. Moreover, the combination of ONYX-015 and chemotherapeutics induced more apoptosis than chemotherapeutics alone in the two colon cancer cell lines independently of their p53 status. We conclude that ONYX-015 virus infection alone or in combination with 5-FU or CPT-11 induced apoptosis in human colon cancer cell lines, independently of p53 status.     

The involvement of p53 in paraquat-induced apoptosis in human lung epithelial-like cells

To investigate the possible role of p53 in the progression of paraquat-induced apoptosis, the authors used two cell lines that were wild-type p53-expressing human lung epithelial-like cell line (L132) and a p53-deficient human promyelocytic leukemia cell line (U937) and explored the linkage between p53, DNA damage, and apoptosis. Following paraquat exposure to L132 cells, the percentage of S-phase cells decreased significantly and the expression of p53 protein increased, suggesting that entry into S phase from G1 phase was blocked. U937 cells showed complete resistance to paraquat, although paraquat-evoked initial single-stranded DNA breaks was shown equally in either L132 or U937 cells, as assessed by single-cell gel electrophoresis. U937 and L132 cells die normally with similar kinetics when exposed to tumor necrosis factor in the presence of cycloheximide, indicating that their capacity to undergo p53-independent mechanisms of inducing apoptosis has an equal rate. These results suggest that paraquat-induced DNA damage caused G1 arrest and apoptosis only in L132 cells, and that p53 protein accumulation is required for the induction of apoptosis by paraquat.     

Induction of apoptosis by the bis-Pt(III) complex [Pt(2)(2-mercaptopyrimidine)(4)Cl(2)

We analyzed both the cytotoxicity and the type of cell death produced by the novel binuclear Pt(III) compound Pt-Spym ([Pt(2)(2-mercaptopyrimidine)(4)Cl(2)]) in kidney human fibroblasts and in human tumor cell lines (HeLa, CH1, CH1cisR and HL-60). The data showed that Pt-Spym displayed higher cytotoxicity against these tumor cells than cisplatin. In contrast, Pt-Spym had low toxicity against normal human fibroblasts. Interestingly, Pt-Spym circumvented cisplatin resistance in CH1cisR cells. We also observed that Pt-Spym induced the characteristic changes attributed to apoptosis in cells with normal levels of p53 protein (CH1 and CH1cisR) and with low levels of p53 protein (HeLa), but not in cells lacking p53 (HL-60). Interestingly, Western blot data indicated that apoptosis induction by Pt-Spym in HeLa, CH1, and CH1cisR cells was not associated with drastic changes in p53 levels. However, cis-DDP strongly decreased p53 levels in CH1 and CH1cisR cells and abolish p53 protein in HeLa cells. Altogether, these results suggest that induction of apoptosis by Pt-Spym requires the presence of p53 protein.     

Korean mistletoe lectin-induced apoptosis in hepatocarcinoma cells is associated with inhibition of telomerase<Emphasis Type="Italic">via</Emphasis> mitochondrial controlled pathway independent of p53

The extract of European mistletoe (Viscum album, L) has been used in adjuvant chemotherapy of cancer and mistletoe lectins are considered to be major active components. The present work was performed to investigate the effects of Korean mistletoe lectin (Viscum album L. coloratum agglutinin, VCA) on proliferation and apoptosis of human hepatoma cells as well as the underlying mechamisms for these effects. We showed that VCA induced apoptosis in both SK-Hep-1 (p53-positive) and Hep 3B (p53-negative) cells through p53- and p21-independent pathways. VCA induced apoptosis by down-regulation of Bcl-2 and by up-regulation of Bax functioning upstream of caspase-3 in both cell lines. In addition, we observed down-regulation of telomerase activity in both VCA-treated cells. Our results provide direct evidence of the anti-tumor potential of this biological response which comes from inhibition of telomerase and consequent inducing apoptosis. VCA-induced apoptosis is regulated by mitochondrial controlled pathway independently of p53. These findings are important for the therapy with preparation of mistletoe because they show that telomerase-dependent mechanism can be targeted by VCA in human hepatocarcinoma. Taken together, our results suggest that the VCA, considered as a telomerase-inhibitor, can be envisaged as a candidate for enhancing sensitivity of conventional anticancer drugs.     

Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis: the role of p53 and p21

Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke-3 cells. However, the presence of oncogenic kRas significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransformed intestinal epithelial cells with inducible expression of kRasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3.     

The labdane diterpene sclareol (labd-14-ene-8, 13-diol) induces apoptosis in human tumor cell lines and suppression of tumor growth in vivo via a p53-independent mechanism of action

The labdane diterpene sclareol has demonstrated significant cytotoxicity against human tumor cell lines and human colon cancer xenografts. Therefore, there is need to elucidate the mode of action of this compound as very little information is known for the anticancer activity of sclareol and other labdane diterpenes, in general. COMPARE analysis of GI(50) values for a number of human cancer cell lines was initially implicated in an effort to assign a putative mechanism of action to the compound. Sclareol-induced cell cycle arrest and apoptosis were assessed by flow cytometry and Western blot analyses. Finally, the anticancer ability of sclareol in vivo was assessed by using human colon cancer xenograft/mouse models. Sclareol arrested in vitro the growth of p53-deficient (HCT116(p53-/-)) human colon cancer cells and subsequently induced apoptosis by activating both caspases-8 and -9. Intraperitoneal administration of liposome-encapsulated sclareol at the maximum tolerated dose induced a marked growth suppression of HCT116(p53-/-) tumors established as xenografts in immunodeficient NOD/SCID mice. In conclusion, we demonstrate herein that sclareol kills human tumor cells by inducing arrest at the G(1)-phase of the cell cycle followed by apoptosis that involves activation of caspases-8, -9 and -3 via a p53-independent mechanism. These findings suggest that liposome-encapsulated sclareol possesses chemotherapeutic potential for the treatment of colorectal and other types of human cancer regardless of the p53-status.     

The effects of NBS1 knockdown by small interfering RNA on the ionizing radiation-induced apoptosis in human lymphoblastoid cells with different p53 status

Mutations of NBS1 are responsible for the human hereditary disease Nijmegen breakage syndrome (NBS), which is characterized by an extremely high cancer rate. In this study, we investigated the influence of NBS1 on ionizing radiation (IR) induced apoptosis. Using small interfering RNA (siRNA) transfection, we knocked down NBS1 protein in three closely related human lymphoblastoid cell lines differing in p53 status: TK6 with a wild-type p53, NH32 with a null mutation of p53, and WTK1 with a mutant p53. We found that up to 48h after 5Gy IR, all three lines showed an obvious induction of apoptosis regardless of the p53 status. The magnitude of apoptosis induction was TK6>NH32>WTK1. This suggested that although p53 is an important modulator of IR-induced apoptosis, other p53-independent apoptosis pathway also exists. Moreover, NBS1 knockdown led to reduction of IR-induced apoptosis in all three lines and both NBS1/ATM/p53/BAX and NBS1/ATM/CHK2/E2F1 apoptosis pathways were partially inactivated. Our results suggest that NBS1 plays an important role in IR-induced apoptosis via both p53-dependent and p53-independent mechanisms. The impaired apoptosis response to DNA damage in NBS1 deficient cells might be one of the important mechanisms of cancer predisposition in NBS patients.     

A profile of the in vitro antitumor activity of lissoclinolide

Lissoclinolide is a small non-nitrogenous lactone isolated from the marine ascidian Lissoclinum patella. Previous studies of lissoclinolide (isolated from a fungus and an actinomycete) have identified varying activity against both Gram-negative and Gram-positive bacteria. In this study, lissoclinolide was able to inhibit cell growth in various mammalian tumor lines at an average IC(50) of 395 nM (determined by MTT conversion after 48-h treatment). Treatment of HCT 116 human colon tumor cells with 2.4 microM lissoclinolide resulted in a strong arrest in the G(2)/M phase of the cell cycle after 24-h exposure. A daughter cell line lacking p53 showed an identical response while there was a slight increase in cytotoxicity towards a p21 null cell line. Although treatment with 2.4 microM lissoclinolide did not result in apoptosis after 48 h, this arrest was not reversible when drug wash out was attempted. The mechanism of action does not appear to involve tubulin, ubiquitin-specific isopeptidases, p53 or p21. COMPARE analysis in the NCI 60 cell line tumor panel revealed a moderate selectivity towards colon tumor cell lines.     

土槿乙酸衍生物PBA-D1诱导Hela细胞凋亡及其机制

目的研究土槿乙酸衍生物PBAD1体外能否诱导宫颈癌细胞株(Hela细胞)发生凋亡及其发生机制。方法采用MTT、SRB、细胞生长曲线、细胞集落、荧光显微镜检测凋亡细胞、DNAladder、流式细胞仪等方法进行凋亡检测,用Westernblot方法检测凋亡过程中相关基因表达的变化。结果土槿乙酸衍生物PBAD1在体外试验中对Hela细胞的杀伤力强,荧光显微镜观察到了凋亡小体;DNAladder法检测到凋亡时DNA降解形成的梯带,流式细胞仪检测到了细胞凋亡峰,同时观察到细胞周期的变化。在凋亡过程中,凋亡相关基因p53基因表达明显增高,而bcl2表达下调。结论PBAD1体外能诱导Hela细胞发生凋亡,其机制可能与p53基因表达上调及bcl2基因表达下调有关。     

Inhibition of G1/S transition potentiates oxaliplatin-induced cell death in colon cancer cell lines

In a series of colorectal cancer cell lines, both necrosis and apoptosis were induced upon exposure to oxaliplatin, and enhanced by co-administration of the Hsp90 inhibitor 17-AAG. We analyzed the effects of these interventions on the cell cycle, and found that oxaliplatin treatment caused G1 and G2 arrest in HCT116 cells, and S-phase accumulation in two p53-deficient cell lines (HT29 and DLD1). Addition of 17-AAG enhanced cell cycle effects of oxaliplatin in HCT116, and induced G1 arrest and decrease in S-phase population in the other cell lines. Analysis of cell cycle proteins revealed that the major difference between the cell lines was that in HCT116, 17-AAG resulted in profound inhibition of expression and phosphorylation of late G1 proteins cyclin E and cdk2, with no effect on p21/WAF1 induction. Consistent with these, an HCT116 p53(-/-) line, lacking p21, showed resistance to oxaliplatin, failure to enter apoptosis, and an accumulation of cells in S-phase. Introduction of p21 in these cells caused reversal of that phenotype, including restoration of the G1 block and re-sensitization to oxaliplatin. Inhibition of G1/S progression using cdk2 inhibitor also enhanced oxaliplatin cytotoxicity. We conclude that in colon cancer cells with impaired p53 function, interventions directed to cycle arrest in G1 may potentiate oxaliplatin activity.     

Peripheral-type benzodiazepine receptor overexpression and knockdown in human breast cancer cells indicate its prominent role in tumor cell proliferation

The peripheral-type benzodiazepine receptor (PBR), an 18-kDa high affinity drug and cholesterol binding protein, is expressed at high levels in various cancers. Its expression is positively correlated with aggressive metastatic behavior in human breast cancer cells. To determine the role of PBR in tumor progression, two human mammary carcinoma cell lines were utilized: the non-aggressive MCF-7 cell line, which expresses extremely low PBR levels, and the highly aggressive MDA-MB-231 cell line, which has much higher PBR levels. We have generated stably transfected lines of the tetracycline-repressible MCF-7 cell line (MCF-7 Tet-Off) with inducible human PBR cDNA. Induction of PBR expression in MCF-7 Tet-Off cells increased PBR ligand binding and cell proliferation. Transfection of MDA-MB-231 cells with multiple siRNAs complementary to PBR (PBR-siRNAs) led to different levels of PBR mRNA knockdown. Lentiviral-mediated PBR RNA interference in MDA-MB-231 cells decreased PBR levels by 50%. Decreased PBR expression was associated with cell cycle arrest at G2 phase, decreased cell proliferation, and significant increases in the protein levels of the cyclin-dependent kinase inhibitor p21(WAF/CIP1). These changes were accompanied by p53 activation seen as increased p53 phosphorylation (Ser15). In parallel, increased proteolytic activation of caspase-3 was also observed. Taken together these results suggest that PBR protein expression is directly involved in regulating cell survival and proliferation in human breast cancer cells by influencing signaling mechanisms involved in cell cycle control and apoptosis.     

Differential apoptosis by indomethacin in gastric epithelial cells through the constitutive expression of wild-type p53 and/or up-regulation of c-myc.

Nonsteroidal anti-inflammatory drug (NSAID)-induced apoptosis is considered to be an important mechanism in the antineoplastic effects and damage produced by the drugs in the gastrointestinal tract. In this study, two different gastric cancer cell lines, MKN28 (mutant-type p53) and AGS (wild-type p53), were compared as to growth inhibition, apoptosis, and cell cycle and apoptosis-related gene expression in response to indomethacin treatment. Cell growth was measured by MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Apoptosis was characterized by acridine orange staining and DNA fragmentation, and cell cycle kinetics by flow cytometry. The mRNA and protein levels of p53, p21waf1/cip1, and c-myc were determined by Northern and Western blotting. The results showed that indomethacin initiated growth inhibition and apoptosis in both cell lines without cell cycle shifting. AGS cells were more sensitive to growth inhibitory activity and apoptosis of indomethacin than MKN28 cells. In MKN28 cells, the levels of p53, p21waf1/cip1, and c-myc mRNA remained unchanged over the 24-hr treatment with indomethacin, but the p53 protein level was elevated after 4 hr. There was no change in the p21waf1/cip1 and c-myc protein levels in the MKN28 cells. In AGS cells, a progressive increase in c-myc mRNA and protein levels was noted, while p53 and p21waf1/cip1 remained unchanged. It can be concluded that wild-type p53 and/or up-regulation of c-myc is associated with indomethacin-mediated differential apoptosis in gastric epithelial cells.     

Human papilloma virus 16 E6 RNA interference enhances cisplatin and death receptor-mediated apoptosis in human cervical carcinoma cells

In cervical cancer, the p53 and retinoblastoma (pRb) tumor suppressor pathways are disrupted by the human papilloma virus (HPV) E6 and E7 oncoproteins, because E6 targets p53 and E7 targets pRb for rapid proteasome-mediated degradation. We have investigated whether E6 suppression with small interfering RNA (siRNA) restores p53 functionality and sensitizes the HPV16-positive cervical cancer cell line SiHa to apoptosis by cisplatin, irradiation, recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), or agonistic anti-Fas antibody. E6 siRNA resulted in decreased E6 mRNA levels and enhanced p53 and p21 expression, demonstrating the restoration of p53 functionality in SiHa cells, without inducing high levels of apoptosis (<10%). Cell surface expression of the proapoptotic death receptors (DRs) DR4, DR5, and Fas was not affected by E6 suppression. E6 suppression conferred susceptibility to cisplatin-induced apoptosis but not to irradiation-, rhTRAIL-, or anti-Fas antibody-induced apoptosis. Combining cisplatin with rhTRAIL or anti-Fas antibody induced even higher apoptosis levels in E6-suppressed cells. At the molecular level, cisplatin treatment resulted in elevated p53 levels, enhanced caspase-3 activation, and reduced p21 levels in E6-suppressed cells. Cisplatin in combination with death receptor ligands enhanced caspase-8 and caspase-3 activation and reduced X-linked inhibitor-of-apoptosis protein (XIAP) levels in these cells. We showed using siRNA that the enhanced apoptosis in E6-supressed cells was related to reduced XIAP levels and not due to reduced p21 levels. In conclusion, targeting E6 or XIAP in combination with cisplatin can efficiently potentiate rhTRAIL-induced apoptosis in HPV-positive cervical cancer cells.