Critical role of CD40-mediated autocrine tumor necrosis factor-alpha in potentiation of cisplatin-induced cytotoxicity in cancer cells.

Activation of CD40, a member of the tumor necrosis factor receptor (TNF-R) family, results in growth inhibition or apoptosis in some tumor cells, making CD40 a potential antitumor therapeutic target. Although it is known that CD40 is able to induce tumor necrosis factor-alpha (TNF-α) secretion and potentiate cisplatin's anticancer activity, whether TNF-α induction is involved in sensitizing cisplatin by CD40 has not been addressed. In this report, we provide evidence substantiating an important role of autocrine TNF-α in potentiation of cisplatin-induced apoptosis by recombinant soluble CD40 ligand (rsCD40L) in different human cancer cell lines. Activation of CD40 by rsCD40L induces two phases of autocrine TNF-α: the rapid early phase involving p38 MAP kinase and the robust and persistent late phase through enhanced tnf-α gene transcription. Blocking TNF-α with either a specific TNFR1 siRNA or a neutralizing anti-TNF-α antibody dramatically attenuated the potentiation effect of rsCD40L on cisplatin-induced cancer cell death. These results reveal an important role of TNF-α induction in CD40's chemosensitization activity and suggest that modulating TNF-α autocrine from cancer cells is an effective option for increasing the anticancer value of chemotherapeutics such as cisplatin.

Crocetin induces cytotoxicity and enhances vincristine-induced cancer cell death via p53-dependent and -independent mechanisms.

AIM: To investigate the anticancer effect of crocetin, a major ingredient in saffron, and its underlying mechanisms. METHODS: Cervical cancer cell line HeLa, non-small cell lung cancer cell line A549 and ovarian cancer cell line SKOV3 were treated with crocetin alone or in combination with vincristine. Cell proliferation was examined using MTT assay. Cell cycle distribution and sub-G(1) fraction were analyzed using flow cytometric analysis after propidium iodide staining. Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit with flow cytometry. Cell death was measured based on the release of lactate dehydrogenase (LDH). The expression levels of p53 and p21(WAF1/Cip1) as well as caspase activation were examined using Western blot analysis. RESULTS: Treatment of the 3 types of cancer cells with crocetin (60-240 µmol/L) for 48 h significantly inhibited their proliferation in a concentration-dependent manner. Crocetin (240 µmol/L) significantly induced cell cycle arrest through p53-dependent and -independent mechanisms accompanied with p21(WAF1/Cip1) induction. Crocetin (120-240 µmol/L) caused cytotoxicity in the 3 types of cancer cells by enhancing apoptosis in a time-dependent manner. In the 3 types of cancer cells, crocetin (60 µmol/L) significantly enhanced the cytotoxicity induced by vincristine (1 µmol/L). Furthermore, this synergistic effect was also detected in the vincristine-resistant breast cancer cell line MCF-7/VCR. CONCLUSION: Ccrocetin is a potential anticancer agent, which may be used as a chemotherapeutic drug or as a chemosensitizer for vincristine.

[Effect of heat shock protein 90 inhibitor 17-dimethylamino-ethylaminogeldanamycin on TNfalpha-mediated apoptosis and TNF-induced NF-kappaB activation].

OBJECTIVE: To investigate the effect of heat shock protein 90 (HSP90) inhibitor 17-dimethylaminoethylaminogeldanamycin (17DMAG) on tumor necrosis factor alpha (TNFalpha) mediated apopotosis in cancer cells and the underlying molecular mechanisms. METHODS: Cell death treated with different concentration of 17DMAG and TNFalpha was detected based on the release of lactate dehydrogenase (LDH) using a cytotoxicity detection kit. For morphological study of cell death, cells were stained with 50 microg/mL of acridine orange and 50 microg/mL of ethidium bromide and observed and photographed under a fluorescence microscope. Activation of apoptosis and NF-kappaB pathway were evaluated on the change of caspase-8, caspase-3, poly (ADP-ribose) polymerase (PARP), receptor-interaction protein (RIP), IkappaB kinase beta (IKKbeta) and inhibitor of IkappaB (IkappaBalpha) by Western blot. RESULTS: 17DMAG sensitized cervical cancer cells HeLa and ovarian cancer cells SKOV3 to TNFalpha-induced cell death in a dose-dependent manner, which was accompanied with degradation of RIP and Ikappakappabeta, and consequent blockage of TNFalpha-induced NFkappaB activation. 17DMAG and TNFalpha cotreated cells showed typical apoptotic morphologies and enhancing of activation of caspases. CONCLUSION: 17DMAG sensitizes cancer cells to TNFalpha-mediated apoptosis through blockage of TNF-induced NF-kappaB activation, and disabling this survival signal with 17DMAG followed by TNF treatment could be an effective new therapeutic strategy for improving the anti-cancer value of TNFalpha.

Reactive oxygen species-mediated apoptosis contributes to chemosensitization effect of saikosaponins on cisplatin-induced cytotoxicity in cancer cells.

BACKGROUND: Saikosaponin-a and -d, two naturally occurring compounds derived from Bupleurum radix, have been shown to exert anti-cancer activity in several cancer cell lines. However, the effect of combination of saikosaponins with chemotherapeutic drugs has never been addressed. Thus, we investigated whether these two saikosaponins have chemosensitization effect on cisplatin-induced cancer cell cytotoxicity. METHODS: Two cervical cancer cell lines, HeLa and Siha, an ovarian cancer cell line, SKOV3, and a non-small cell lung cancer cell line, A549, were treated with saikosaponins or cisplatin individually or in combination. Cell death was quantitatively detected by the release of lactate dehydrogenase (LDH) using a cytotoxicity detection kit. Cellular ROS was analyzed by flow cytometry. Apoptosis was evaluated by AO/EB staining, flow cytometry after Anexin V and PI staining, and Western blot for caspase activation. ROS scavengers and caspase inhibitor were used to determine the roles of ROS and apoptosis in the effects of saikosaponins on cisplatin-induced cell death. RESULTS: Both saikosaponin-a and -d sensitized cancer cells to cisplatin-induced cell death in a dose-dependent manner, which was accompanied with induction of reactive oxygen species (ROS) accumulation. The dead cells showed typical apoptotic morphologies. Both early apoptotic and late apoptotic cells detected by flow cytometry were increased in saikosaponins and cisplatin cotreated cells, accompanied by activation of the caspase pathway. The pan-caspase inhibitor z-VAD and ROS scavengers butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC) dramatically suppressed the potentiated cytotoxicity achieved by combination of saikosaponin-a or -d and cisplatin. CONCLUSIONS: These results suggest that saikosaponins sensitize cancer cells to cisplatin through ROS-mediated apoptosis, and the combination of saikosaponins with cisplatin could be an effective therapeutic strategy.

Absence of association between CD86 +1057G/A polymorphism and coronary artery disease.

CD86, one of the key costimulatory molecules, is not only involved in the initiation of T-cell immunity but also plays important roles in the development of cardiovascular diseases. The purpose of this study was to investigate the association between the CD86 polymorphism and the risk of coronary artery disease (CAD) in a Chinese population. We analyzed single-nucleotide polymorphism of CD86 +1057G/A (rs1129055) in 164 patients with CAD and 299 healthy controls by performing polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing assay. No significant association was observed in the genotype and allele frequencies of +1057G/A polymorphism between cases and controls, indicating that CD86 +1057G/A polymorphism may not be associated with CAD in the Chinese population.

Concurrent blockade of the NF-kappaB and Akt pathways potently sensitizes cancer cells to chemotherapeutic-induced cytotoxicity.

Nuclear factor-kappaB (NF-kappaB) and Akt are two major cell survival pathways that are often constitutively activated and can be further stimulated by chemotherpeutics in cancer cells. Although individually targeting the NF-kappaB or Akt has been reported to sensitize caner therapy, the effectiveness of concurrent blocking these two pathways for chemosensitizing of cancer cells to genotoxic therapeutics has not been investigated. In the present study, we investigate the activation of the NF-kappaB and Akt pathways by two frontline anticancer drugs cisplatin and etopside in a variety of cancer cell lines. The effects of blocking these two survival pathways individually or concurrently on cisplatin- or etopside-induced cytotoxicity were detected. The results show that cisplatin and etopside activate both NF-kappaB and Akt in cancer cells. Blockade of either of these pathways with chemical inhibitors or siRNA moderately sensitized cancer cells to cisplatin- or etopside-induced cytotoxicity. Strikingly, much more effective potentiation of cytotoxicity to these anticancer drugs was achieved when NF-kappaB and Akt were concurrently blocked. These data suggest that NF-kappaB and Akt cooperatively attenuate therapeutic-induced cytotoxicity and concurrently blocking these pathways is an effective strategy for improving the anticancer efficacy of therapeutics.