Bak compensated for Bax in p53-null cells to release cytochrome c for the initiation of mitochondrial signaling during Withanolide D-induced apoptosis

The goal of cancer chemotherapy to induce multi-directional apoptosis as targeting a single pathway is unable to decrease all the downstream effect arises from crosstalk. Present study reports that Withanolide D (WithaD), a steroidal lactone isolated from Withania somnifera, induced cellular apoptosis in which mitochondria and p53 were intricately involved. In MOLT-3 and HCT116p53+/+ cells, WithaD induced crosstalk between intrinsic and extrinsic signaling through Bid, whereas in K562 and HCT116p53-/- cells, only intrinsic pathway was activated where Bid remain unaltered. WithaD showed pronounced activation of p53 in cancer cells. Moreover, lowered apoptogenic effect of HCT116p53-/- over HCT116p53+/+ established a strong correlation between WithaD-mediated apoptosis and p53. WithaD induced Bax and Bak upregulation in HCT116p53+/+, whereas increase only Bak expression in HCT116p53-/- cells, which was coordinated with augmented p53 expression. p53 inhibition substantially reduced Bax level and failed to inhibit Bak upregulation in HCT116p53+/+ cells confirming p53-dependent Bax and p53-independent Bak activation. Additionally, in HCT116p53+/+ cells, combined loss of Bax and Bak (HCT116Bax-Bak-) reduced WithaD-induced apoptosis and completely blocked cytochrome c release whereas single loss of Bax or Bak (HCT116Bax-Bak+/HCT116Bax+Bak-) was only marginally effective after WithaD treatment. In HCT116p53-/- cells, though Bax translocation to mitochondria was abrogated, Bak oligomerization helped the cells to release cytochrome c even before the disruption of mitochondrial membrane potential. WithaD also showed in vitro growth-inhibitory activity against an array of p53 wild type and null cancer cells and K562 xenograft in vivo. Taken together, WithaD elicited apoptosis in malignant cells through Bax/Bak dependent pathway in p53-wild type cells, whereas Bak compensated against loss of Bax in p53-null cells.     

Polo-like kinase 1 inhibitors,mitotic stress and the tumor suppressor p53

Polo-like kinase 1 has been established as one of the most attractive targets for molecular cancer therapy. In fact, multiple small-molecule inhibitors targeting this kinase have been developed and intensively investigated. Recently, it has been reported that the cytotoxicity induced by Plk1 inhibition is elevated in cancer cells with inactive p53, leading to the hypothesis that inactive p53 is a predictive marker for the response of Plk1 inhibition. In our previous study based on different cancer cell lines, we showed that cancer cells with wild type p53 were more sensitive to Plk1 inhibition by inducing more apoptosis, compared with cancer cells depleted of p53. In the present work, we further demonstrate that in the presence of mitotic stress induced by different agents, Plk1 inhibitors strongly induced apoptosis in HCT116 p53^+/+ cells, whereas HCT116 p53^−/− cells arrested in mitosis with less apoptosis. Depletion of p53 in HCT116 p53^+/+ or U2OS cells reduced the induction of apoptosis. Moreover, the surviving HCT116 p53^−/− cells showed DNA damage and a strong capability of colony formation. Plk1 inhibition in combination with other anti-mitotic agents inhibited proliferation of tumor cells more strongly than Plk1 inhibition alone. Taken together, the data underscore that functional p53 strengthens the efficacy of Plk1 inhibition alone or in combination by strongly activating cell death signaling pathways. Further studies are required to investigate if the long-term outcomes of losing p53, such as low differential grade of tumor cells or defective DNA damage checkpoint, are responsible for the cytotoxicity of Plk1 inhibition.     

Stabilization and translocation of p53 to mitochondria is linked to Bax translocation to mitochondria in simvastatin-induced apoptosis

Statins are cholesterol-lowing drugs with pleiotropic effects including cytotoxicity to cancer cells. In this study, we investigated the signaling pathways leading to apoptosis by simvastatin. Simvastatin induced cardinal features of apoptosis including increased DNA fragmentation, disruption of mitochondrial membrane potential (MMP), and increased caspase-3 activity by depleting isoprenoids in MethA fibrosarcoma cells. Interestingly, the simvastatin-induced apoptosis was accompanied by p53 stabilization involving Mdm2 degradation. The apoptosis was ameliorated in p53 knockdown clones of MethA cells as well as p53^−/− HCT116 cells. The stabilized p53 protein translocated to mitochondria with Bax, and cytochrome c was released into cytosol. Moreover, knockdown or deficiency of p53 expression reduced both Bax translocation to mitochondria and MMP disruption in simvastatin-induced apoptosis. Taken together, these all indicate that stabilization and translocation of p53 to mitochondria is involved in Bax translocation to mitochondria in simvastatin-induced apoptosis.     

Photodynamic therapy-induced death of HCT 116 cells: Apoptosis with or without Bax expression

Cell death following photodynamic therapy (PDT) with the photosensitizer Pc 4 involves the intrinsic pathway of apoptosis. To evaluate the importance of Bax in apoptosis after PDT, we compared the PDT responses of Bax-proficient (Bax^+/−) and Bax knock-out (BaxKO) HCT116 human colon cancer cells. PDT induced a slow apoptotic process in HCT Bax^+/− cells following a long delay in the activation of Bax and release of cytochrome c from mitochondria. Although cytochrome c was not released from mitochondria following PDT in BaxKO cells, an alternative mechanism of caspase-dependent apoptosis with extensive chromatin and DNA degradation was found in these cells. This alternative process was less efficient and slower than the normal apoptotic process observed in Bax^+/− cells. Early events upon PDT, such as the loss of mitochondrial membrane potential, photodamage to Bcl-2, and activation of p38 MAP kinase, were observed in both HCT116 cell lines. In spite of differences in the efficiency and mode of apoptosis induced by PDT in the Bax^+/− and BaxKO cells, they were found to be equally sensitive to killing by PDT, as determined by loss of clonogenicity. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to and independent of Bax activation, but the process of cellular disassembly differs in Bax-expressing vs. non-expressing cells.     

Triad 1 induces apoptosis by p53 activation

Triad 1 (2 RING [really interesting new gene] fingers and DRIL [double RING finger linked] 1) is an E3 ligase that induces apoptosis and clonogenic inhibition in myeloid cells through Gfi-1 stabilization. Here we demonstrate that Triad 1 induces apoptosis in several cancer cell lines including MCF7, A549, U2OS, and HCT 116 p53^+/+ cells via its RING ligase activity. Interestingly, in these cancer cells, Triad 1-induced apoptosis is not mediated by Gfi-1 stabilization but is instead p53-dependent. Moreover, Triad 1 promotes transactivation of p53. These results suggest that Triad 1 can induce apoptosis through its ligase activity via p53 activation.     

Trisubstituted and tetrasubstituted pyrazolines as a novel class of cell-growth inhibitors in tumor cells with wild type p53

Derivatives with scaffolds of 1,3,5-tri-substituted pyrazoline and 1,3,4,5-tetra-substituted pyrazoline were synthesized and tested for their inhibitory effects versus the p53^+/+ HCT116 and p53^?/? H1299 human tumor cell lines. Several compounds were active against the two cell lines displaying IC₅₀ values in the low micromolar range with a clearly more pronounced effect on the p53^+/+ HCT116 cells. The compound class shows excellent developability due to the modular synthesis, allowing independent optimization of all three to four key substituents to improve the properties of the molecules.     

The relative contribution of pro-apoptotic p53-target genes in the triggering of apoptosis following DNA damage in vitro and in vivo

The p53 pathway displays a large degree of redundancy in the expression of a number of pro-apoptotic mechanisms following DNA damage that, among others, involves increased expression of several pro-apoptotic genes through transactivation. Spatial and temporal cellular contexts contribute to the complexity of the regulation of apoptosis, hence different genes may show a cell- and tissue-dependent specificity with regard to the regulation of cell death and act in concert or show redundancy with one and another. We used siRNA technology to assess the effect of multiple ablations of documented pro-apoptotic p53 target genes (PPG) in the colorectal cancer cell line HCT116 and generated mice deficient in both of the extrinsic and intrinsic PPGs genes Dr5 and Puma following treatment with chemotherapeutics and ionizing radiation. DR5, Fas, Bax, Bad, Puma and Bnip3L were induced by 5-FU and adriamycin (ADR) in HCT116 cells in a p53-dependent manner. The resulting caspase 3/7 activity in HCT116 cells following treatment were suppressed by ablated expression of the PPGs in the extrinsic as well as the intrinsic pathway. To our surprise, knocking-down any of the PPGs concomitantly with DR5 did not further inhibit caspase 3/7 activity whereas inhibiting DR5-expression in HCT116Bax knockdown (kd) and HCT116Fas kd did, suggesting that these genes act downstream or in synergy with DR5. This was supported by our in vivo observations, since Puma and Dr5 were equally efficient in protecting cells of the spleen from sub-lethal radiation-induced apoptosis but less effective compared with irradiated p53^-/- mice. To our surprise, Dr5^-/-; Puma^-/- mice did not show additive protection from radiation-induced apoptosis in any of the investigated organs. Our data indicates that the intrinsic pathway may rely on extrinsic signals to promote cell death in a cell- and tissue-dependent manner following DNA damage. Furthermore, p53 must rely on mechanisms independent of DR5 and PUMA to initiate apoptosis following γ-radiation in the spleen and thymus in vivo.     

Design,Synthesis and In Vitro Activity of Anticancer Styrylquinolines. The p53 Independent Mechanism of Action

A group of styrylquinolines were synthesized and tested for their anti-proliferative activity. Anti-proliferative activity was evaluated against the human colon carcinoma cell lines that had a normal expression of the p53 protein (HCT116 p53^+/+) and mutants with a disabled TP53 gene (HCT116 p53^-/-) and against the GM 07492 normal human fibroblast cell line. A SAR study revealed the importance of Cl and OH as substituents in the styryl moiety. Several of the compounds that were tested were found to have a marked anti-proliferative activity that was similar to or better than doxorubicin and were more active against the p53 null than the wild type cells. The cellular localization tests and caspase activity assays suggest a mechanism of action through the mitochondrial pathway of apoptosis in a p53-independent manner. The activity of the styrylquinoline compounds may be associated with their DNA intercalating ability.     

The relative contribution of pro-apoptotic p53 target genes in the triggering of apoptosis following DNA damage in vitro and in vivo

The p53 pathway displays a large degree of redundancy in the expression of a number of pro-apoptotic mechanisms following DNA damage that, among others, involves increased expression of several pro-apoptotic genes through transactivation. Spatial and temporal cellular contexts contribute to the complexity of the regulation of apoptosis, hence different genes may show a cell- and tissue-dependent specificity with regard to the regulation of cell death and act in concert or show redundancy with one and another. We used siRNA technology to assess the effect of multiple ablations of documented pro-apoptotic p53 target genes (PPG) in the colorectal cancer cell line HCT116 and generated mice deficient in both of the extrinsic and intrinsic PPGs genes Dr5 and Puma following treatment with chemotherapeutics and ionizing radiation. DR5, Fas, Bax, Bad, Puma and Bnip3L were induced by 5-FU and adriamycin (ADR) in HCT116 cells in a p53-dependent manner. The resulting caspase 3/7 activity in HCT116 cells following treatment were suppressed by ablated expression of the PPGs in the extrinsic as well as the intrinsic pathway. To our surprise, knocking-down any of the PPGs concomitantly with DR5 did not further inhibit caspase 3/7 activity whereas inhibiting DR5-expression in HCT116Bax knockdown (kd) and HCT116Fas kd did, suggesting that these genes act downstream or in synergy with DR5. This was supported by our in vivo observations, since Puma and Dr5 were equally efficient in protecting cells of the spleen from sub-lethal radiation-induced apoptosis but less effective compared with irradiated p53^−/− mice. To our surprise, Dr5^−/−; Puma^−/− mice did not show additive protection from radiation-induced apoptosis in any of the investigated organs. Our data indicates that the intrinsic pathway may rely on extrinsic signals to promote cell death in a cell- and tissue-dependent manner following DNA damage. Furthermore, p53 must rely on mechanisms independent of DR5 and PUMA to initiate apoptosis following γ-radiation in the spleen and thymus in vivo.Key words: p53, KILLER/DR5, PUMA, apoptosis, DNA damage     

Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53^+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53^−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53^−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53.     

Radiosensitization of wildtype p53 cancer cells by the MDM2-inhibitor PXN727 is associated with altered heat shock protein 70 (Hsp70) levels

The oncoprotein MDM2 (murine double minute 2) is often overexpressed in human tumors and thereby attenuates the function of the tumor suppressor p53. In this study, we investigated the effects of the novel MDM2-inhibitor PXN727 on p53 activation, cell proliferation, cell cycle distribution and radiosensitivity. Since the localization of heat shock protein 70 (Hsp70) exerts different effects on radioresistance of tumor cells, we investigated the impact of PXN727 on intracellular, membrane, and secreted Hsp70 levels. We could show that PXN727 exerts its effects on wildtype p53 (HCT116 p53^+/+, A549) but not p53 depleted (HCT116 p53^−/−) or mutated (FaDu) tumor cells. PXN727 activates p53, induces the expression of p21, reduces the proportion of cells in the radioresistant S-phase and induces senescence. Radiosensitivity was significantly increased by PXN727 in HCT116 p53^+/+ tumor cells. Furthermore, PXN727 causes a downregulation of Hsp70 membrane expression and an upregulated secretion of Hsp70 in wildtype p53 tumor cells. Our data suggest that re-activation of p53 by MDM2-inhibition modulates Hsp70 membrane expression and secretion which might contribute to the radiosensitizing effect of the MDM2-inhibitor PXN727.     

Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53

Drug resistance to 5-fluorouracil (5-FU) is still a major limitation to its clinical use. In addition, the clinical value of p53 as a predictive marker for 5-FU-based chemotherapy remains a matter of debate. Here, we used HCT116 human colorectal cancer cells expressing wild-type p53 and investigated whether inhibition of Fas expression by interference RNA modulates 5-FU-induced apoptosis. Cells were treated with 5-FU (1, 4 or 8 microM) for 8-48 h. Cell viability was evaluated by trypan blue dye exclusion. Apoptosis was assessed by changes in nuclear morphology and caspase activity. The interference RNA technology was used to silence Fas expression. Caspase activation, p53, Fas, cytochrome c, and Bcl-2 family protein expression was evaluated by immunoblotting. 5-FU was cytotoxic in HCT116 cells (p<0.001). Nuclear fragmentation and caspase-3, -8 and -9 activities were also markedly increased in HCT116 cells after 5-FU (p<0.001). In addition, wild-type p53 and Fas expression were 25- and 4-fold increased (p<0.05). Notably, when interference RNA was used to inhibit Fas, 5-FU-mediated nuclear fragmentation and caspase activity were markedly reduced in HCT116 cells. Finally, western blot analysis of mitochondrial extracts from HCT116 cells exposed to 5-FU showed a 6-fold increase in Bax, together with a 3-fold decrease in cytochrome c (p<0.001). In conclusion, 5-FU exerts its cytotoxic effects, in part, through a p53/Fas-dependent apoptotic pathway that involves Bax translocation and mitochondrial permeabilization.     

Ganoderic acid Me induces G1 arrest in wild-type p53 human tumor cells while G1/S transition arrest in p53-null cells

The mechanism of cell cycle arrest of tumor cells induced by ganoderic acid Me (GA-Me) is not understood. In this work, GA-Me was found to possess remarkable cytotoxicity on highly metastatic lung carcinoma 95-D cell line in both dose- and time-dependent manners. The effect of GA-Me on cell cycle arrest was found in 95-D, p53-null lung cancer cells H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? human colon cancer cells. To obtain an insight into the role of p53 in cell cycle arrest by GA-Me, 95-D, H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? cells were used for further investigation. GA-Me arrested cell cycle at G₁ phase in 95-D and HCT-116 p53^+/+ cells while S phase or G₁/S transition arrest in H1299 and HCT-116 p53^?/? cells. The results suggested that p53 may be a target of GA-Me, and it may be looked at as a new promising candidate for the treatment of carcinoma cells.     

Carbon-Ion Beam Irradiation Kills X-Ray-Resistant p53-Null Cancer Cells by Inducing Mitotic Catastrophe

Background and Purpose

To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies.

Materials and Methods

DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53^+/+ and p53^-/-, respectively) were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA double-strand breaks (DSBs) by immunostaining of phosphorylated H2AX (γH2AX), and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3.

Results

The p53^-/- cells were more resistant than the p53^+/+ cells to X-ray irradiation, while the sensitivities of the p53^+/+ and p53^-/- cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53^+/+ cells but not the p53^-/- cells. In the p53^-/- cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring long-retained DSBs at 24 h post-irradiation.

Conclusions

Efficient induction of mitotic catastrophe in apoptosis-resistant p53-deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment.     

Anti-cancer effects of JKA97 are associated with its induction of cell apoptosis via a Bax-dependent and p53-independent pathway

p53, one of the most commonly mutated genes in human cancers, is thought to be associated with cancer development. Hence, screening and identifying natural or synthetic compounds with anti-cancer activity via p53-independent pathway is one of the most challenging tasks for scientists in this field. Compound JKA97 (methoxy-1-styryl-9H-pyrid-[3,4-b]-indole) is a small molecule synthetic anti-cancer agent, with unknown mechanism(s). In this study we have demonstrated that the anti-cancer activity of JKA97 is associated with apoptotic induction via p53-independent mechanisms. We found that co-incubation of human colon cancer HCT116 cells with JKA97 inhibited HCT116 cell anchorage-independent growth in vitro and tumorigenicity in nude mice and also induced a cell apoptotic response, both in the cell culture model and in a tumorigenesis nude mouse model. Further studies showed that JKA97-induced apoptosis was dramatically impaired in Bax knock-out (Bax(-/-)) HCT116 cells, whereas the knock-out of p53 or PUMA did not show any inhibitory effects. The p53-independent apoptotic induction by JKA97 was confirmed in other colon cancer and hepatocarcinoma cell lines. In addition, our results showed an induction of Bax translocation and cytochrome c release from the mitochondria to the cytosol in HCT116 cells, demonstrating that the compound induces apoptosis through a Bax-initiated mitochondria-dependent pathway. These studies provide a molecular basis for the therapeutic application of JKA97 against human cancers with p53 mutations.