Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors: Evidence for the role of c-Jun N-terminal kinase activation

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53−/−) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.     

The cyclin-dependent kinase inhibitor butyrolactone is a potent inhibitor of p21 (WAF1/CIP1 expression)

Butyrolactone I (BL) is a competitive inhibitor of ATP for binding and activation of cyclin-dependent kinases and is a potent inhibitor of cell cycle progression. Treatment of H460 human lung and SW480 human colon cancer cells with doses of BL that exceed the Ki for CDK inhibition but which are much lower than doses required to inhibit MAPK, PKA, PKC, or EGFR lead to a rapid significant reduction of endogenous p21 protein expression. BL-dependent inhibition of p21 expression appears to be p53-independent. BL-dependent p21 degradation was blocked by lactacystin, consistent with the hypothesis that there is accelerated p21 proteasomal degradation in the presence of BL. BL also inhibited the p53-dependent increase of p21 protein expression in cells exposed to the DNA damag-ing agent etoposide, and favored a greater G2/M arrest as compared to the non-BL exposed cells. BL accelerated the degradation of exogenously expressed p21 that was not observed with a C-terminal truncated form of p21. Degradation of exogenous p21 led to a shift to G2 accumulation in the cells exposed to BL. We conclude that BL has effects on the cell cycle beyond its role as a CDK inhibitor and can be used as a novel tool to study the mechanism of p21 degradation and the consequences towards p21- dependent checkpoints.     

Heat shock protects HCT116 and H460 cells from TRAIL-induced apoptosis

Heat shock proteins have been shown to protect cells from a variety of stressful conditions, including hyperthermia, oxidative and DNA damage, serum withdrawal, and a variety of chemicals. HSP27, HSP70, and HSP90 have been shown to downregulate different aspects of apoptosome assembly. TRAIL is a member of the TNF family of ligands and is a promising anti-cancer agent. It has been shown to be nontoxic to most normal cell types, while it is a potent killer of many different cancer cells. TRAIL engages both the receptor-mediated (extrinsic) and the mitochondria-initiated (intrinsic) cascades. We tested whether heat shock affects TRAIL-induced apoptosis in different cancer cells. TRAIL treatment does not induce HSP27, HSP70, or HSP90 levels. Nonetheless, when treated with TRAIL for 3 h after release from heat shock, the human colon cancer cell line HCT116 is protected from apoptosis whereas the human colon cancer cell line SW480 is not. This pattern is consistent with the previously observed behavior of HCT116 as Type II cells that depend on mitochondrial signaling and SW480 as Type I, whose TRAIL-induced death is not sensitive to inhibition of caspase 9. Moreover, the failure of heat shock to protect SW480 cells is not due to a lack of HSP70 or HSP90 upregulation. HSP70 and HSP90 are induced 3 h after release from heat shock, whereas HSP27 is induced much later. Thus, the observed protective effect against TRAIL is probably due to the anti-apoptotic effects of HSP70 and HSP90. These results further illustrate interactions between TRAIL receptor signaling and the intrinsic cell death pathway and have practical implications for the potential use of TRAIL and hyperthermia in cancer therapy.     

Hybrid origin of Japanese mice "Mus musculus molossinus": evidence from restriction analysis of mitochondrial DNA

The Japanese mouse, Mus musculus molossinus, has long been considered an independent subspecies of the house mouse. A survey of restriction- site haplotypes of mitochondrial DNA (mtDNA) showed that Japanese mice have two main maternal lineages. The most common haplotype is closely related to the mtDNA of the European subspecies M. m. musculus. The other common haplotype and two minor ones are closely related to each other and to the mtDNA of an Asiatic subspecies, M. m. castaneus. Two other rare variants are probably the result of recent contamination by European M. m. domesticus. The musculus type of mtDNA is found in the southern two-thirds of Japan, whereas the common castaneus type is found in the northern third and the minor variants are found sporadically throughout Japan. The castaneus mtDNA lineage had a few minor variants, whereas the musculus lineage was completely monomorphic. By contrast, the native population of M. m. castaneus and the Chinese and Korean musculus populations were highly polymorphic. These results suggest that M. m. molossinus is a hybrid between ancestral colonies, possibly very small, of M. m. musculus and M. m. castaneus, rather than an independent subspecies.