Radiosensitizing effects of plumbagin in cervical cancer cells is through modulation of apoptotic pathway

Radiotherapy is the primary line of cancer treatment for cervical cancer and is known to induce cell death in tumors. Radiotherapy is however limited by the total dose that can be given without damaging normal tissue. Plumbagin, a naturally occurring naphthaquinone, has been reported to have free radical producing properties. Hence we hypothesized that plumbagin could also have properties that could modify effects of radiation on cervical cancer cells. Radiation in combination with plumbagin may thus have treatment augmenting effects. Results from our studies have shown that a lower dose of radiation in combination with plumbagin could induce apoptosis more effectively compared to a higher dose of radiation alone. Plumbagin in combination with 2 Gy of radiation was very effective in inducing apoptosis, when compared to a higher radiation dose of 10 Gy alone. This combination also showed a fivefold increase in the activation of caspase 3 in C33A cells. Activation of effector caspases confirms that the induction of apoptosis by irradiation and plumbagin involves caspase-dependent pathways. Expression of apoptotic regulatory molecules Bcl-2, Bax and Survivin was also modulated by plumbagin in combination with radiation. In summary, this study shows that a combination of plumbagin and radiation augmented cell growth inhibition compared to higher radiation dose alone, thus indicating that plumbagin may be a potential radiosensitizer acting through the induction of apoptosis.     

Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen receptor dependent modulation of the apoptotic pathway

Despite recent advances in diagnosis and management, prostrate cancer remains the second most common cause of death from cancer in American men, after lung cancer. Failure of chemotherapies and hormone-deprivation therapies is the major cause of death in patients with castration-resistant prostate cancer (CRPC). Currently, the androgen inhibitors enzalutamide and abiraterone are approved for treatment of metastatic CRPC. Here we show for the first time that both enzalutamide and abiraterone render prostate tumor cells more sensitive to T cell-mediated lysis through immunogenic modulation, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, the NAIP gene was significantly down-regulated in human prostate tumor cells treated in vitro and in vivo with enzalutamide. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of enzalutamide and abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, especially for patients with minimal response to enzalutamide or abiraterone alone, or for patients who have developed resistance to ADT.     

miR‐106a is frequently upregulated in gastric cancer and inhibits the extrinsic apoptotic pathway by targeting FAS

Emerging evidence has shown the association of aberrantly expressed miR‐106a with cancer development, however, little is known about its potential role in gastric carcinogenesis. In our present study, obviously overexpressed miR‐106a was found in gastric cancer tissues compared with their nontumor counterparts. Suppression of miR‐106a significantly inhibited gastric cancer cell proliferation and triggered apoptosis. Bioinformatic analysis combining with validation experiments identified FAS as a direct target of miR‐106a. Rescue experiments and examination of caspase‐8, PARP and caspase‐3 further approved that miR‐106a could inhibit gastric cancer cell apoptosis through interfering with FAS‐mediated apoptotic pathway. Moreover, a significant inverse correlation was found between miR‐106a and FAS expression not only in gastric cancer cell lines but also in gastric cancer specimens. Taken together, these findings suggest that ectopicly overexpressed miR‐106a may play an oncogenic role in gastric carcinogenesis and impair extrinsic apoptotic pathway through targeting FAS. © 2012 Wiley Periodicals, Inc.     

Fhit modulation of the Akt-survivin pathway in lung cancer cells: Fhit-tyrosine 114 (Y114) is essential

The Fhit tumor suppressor binds and hydrolyses diadenosine polyphosphates and the Fhit-substrate complex has been proposed as a proapoptotic effector, as determined by infection of susceptible cancer cells with adenoviruses carrying wild-type fragile histidine triad (FHIT) or catalytic site mutants. The highly conserved Fhit tyrosine 114 (Y114), within the unstructured loop C-terminal of the catalytic site, can be phosphorylated by Src family tyrosine kinases, although endogenous phospho-Fhit is rarely detected. To explore the importance of Y114 and identify Fhit-mediated signaling events, wild-type and Y114 mutant FHIT-expressing adenoviruses were introduced into two human lung cancer cell lines. Caspase-dependent apoptosis was effectively induced only by wild-type but not Y114 mutant Fhit proteins. By expression profiling of FHIT versus mutant FHIT-infected cells, we found that survivin, an Inhibitor of Apoptosis Protein (IAP) family member, was significantly decreased by wild-type Fhit. In addition, Fhit inhibited activity of Akt, a key effector in the phosphatidylinositol 3-OH kinase (PI3K) pathway; loss of endogenous Fhit expression caused increased Akt activity in vitro and in vivo, and overexpression of constitutively active Akt inhibited Fhit-induced apoptosis. The results indicate that the Fhit Y114 residue plays a critical role in Fhit-induced apoptosis, occurring through inactivation of the PI3K-Akt-survivin signal pathway.     

Amiloride augments TRAIL-induced apoptotic death by inhibiting phosphorylation of kinases and phosphatases associated with the P13K-Akt pathway

We have previously shown that low extracellular pH (pHe) promotes cell killing by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we examined whether amiloride, an inhibitor of the Na(+)/H(+) antiporter capable of lowering the intracellular pH (pHi), can potentiate TRAIL-induced apoptotic death. Human prostate adenocarcinoma DU-145 cells were treated with various concentrations of TRAIL (10-200 ng/ml) and/or amiloride (0.1-1 mM) for 4 h. Amiloride, which caused little or no cytotoxicity by itself, enhanced TRAIL-induced apoptosis. The TRAIL-mediated activation of caspase, and PARP (poly (ADP-ribose) polymerase) cleavage were both promoted by amiloride. Western blot analysis showed that combined treatment with TRAIL and amiloride did not change the levels of TRAIL receptors (death receptor (DR)4, DR5, and DcR2 (decoy recepter 2) or antiapoptotic proteins (FLICE-inhibitory protein (FLIP), inhibitor of apoptosis (IAP), and Bcl-2). However, unlike pHe, amiloride promoted the dephosphorylation of Akt. Interestingly, amiloride also induced the dephosphorylation of P13K (phosphatidylinositol 3-kinase) and PDK-1 (phosphoinositide-dependent kinase-1) kinases along with PTEN (phosphatase and tensin homolog deleted on chromosome 10) and PP1alpha phosphatases. In vitro kinase assays revealed that amiloride inhibited phosphorylation of kinases and phosphatases by competing with ATP. Taken together, the present studies suggest that amiloride enhances TRAIL-induced cytotoxicity by inhibiting phosphorylation of the PI3K-Akt pathway-associated kinases and phosphatases.     

Pharmacokinetics of cis-dichloro-trans-dihydroxy-bis-isopropylamine platinum IV (CHIP) in patients with advanced cancer

Summary The pharmacokinetics of a second-generation platinum (Pt) analog cis-dichloro-trans-dihydroxy-bis-isopropylamine platinum IV (CHIP) have been studied in 12 patients at doses from 20 to 350 mg/m². Three Pt species have been measured: total Pt and non-protein-bound Pt by atomic absorption spectrophotometry, and unchanged CHIP by separation on high-performance liquid chromatography followed by atomic absorption spectrophotometry. Plasma decay of total Pt was biexponential at all doses with a -phase half-life of 32.1–124 h. Plasma decay of filterable Pt was monoexponential at low doses but biexponential at high doses, with a terminal-phase half-life of 17.8–54.6 h. Plasma decay of unchanged CHIP was monoexponential at all doses, with a half-life of 0.64–1.27 h. Excretion of Pt after CHIP was rapid up to 10 h after the end of infusion and then slow. The total recovery of Pt was 15%–61% of the dose at 24 h in 19 patients. The data indicated that essentially all plasma Pt after 12 h is in the form of metabolites, most of which are protein-bound. The most striking difference between CHIP and reported data for cisplatin is the biexponential decay of non-protein-bound Pt.     

Vitamin E succinate in combination with mda-7 results in enhanced human ovarian tumor cell killing through modulation of extrinsic and intrinsic apoptotic pathways

Adenovirus-mediated mda-7 (Ad-mda7) gene transfer has been shown to induce apoptosis in various human cancer cells while sparing normal cells. Vitamin E succinate (VES) is also known to exhibit antitumor activity against a number of human cancer cell lines. We hypothesized that a combination of the two agents would produce an enhanced antitumor effect in MDAH2774 human ovarian cancer cells. Treatment of MDAH2774 cells with Ad-mda7 plus VES resulted in enhanced antitumor activity that involved the activation of two apoptotic pathways. Activation of the extrinsic pathway was demonstrated by increased cell-surface Fas expression and cleavage of Bid and caspase-8. Activation of the intrinsic pathway was demonstrated by disruption of mitochondrial potential; and activation of downstream capase-9 and caspase-3 via cytochrome C release. In contrast, the combination of Ad-mda7 plus VES did not show any antitumor activity against normal fibroblasts, indicating selective tumor cell killing. Our in vitro results provide a basis for further preclinical testing of Ad-mda7 plus VES as a potential cancer treatment strategy.     

The small heat shock protein HspB2 is a novel anti-apoptotic protein that inhibits apical caspase activation in the extrinsic apoptotic pathway

Members of the conserved small heat shock protein (sHSP) family, such as αB-crystallin and Hsp27, are constitutively expressed in diverse malignancies and have been linked to several hallmark features of cancer including apoptosis resistance. In contrast, the sHSP HspB2/MKBP, which shares an intergenic promoter with αB-crystallin, was discovered as a chaperone of the myotonic dystrophy protein kinase and has not been previously implicated in apoptosis regulation. Here we describe a new function for HspB2 as a novel inhibitor of apical caspase activation in the extrinsic apoptotic pathway. Specifically, we demonstrate that HspB2 is expressed in a subset of human breast cancer cell lines and that ectopic expression of HspB2 in breast cancer cells confers resistance to apoptosis induced by both TRAIL and TNF-α. We also show that HspB2 inhibits the extrinsic apoptotic pathway by suppressing apical caspases-8 and 10 activation, thereby blocking downstream apoptotic events, such as Bid cleavage and caspase-3 activation. Consistent with these in vitro effects, HspB2 attenuates the anti-tumor activity of TRAIL in an orthotopic xenograft model of breast cancer. Collectively, our results reveal a novel function of HspB2 as an anti-apoptotic protein that negatively regulates apical caspase activation in the extrinsic apoptotic pathway.     

P27(kip1) down-regulation is associated with trastuzumab resistance in breast cancer cells

Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody directed against HER-2. The objective response rate to trastuzumab monotherapy is 12-34% for a median duration of 9 months, by which point most patients become resistant to treatment. We created two trastuzumab-resistant (TR) pools from the SKBR3 HER-2-overexpressing breast cancer cell line to study the mechanisms by which breast cancer cells escape trastuzumab-mediated growth inhibition. Both pools maintained her-2 gene amplification and protein overexpression. Resistant cells demonstrated a higher S-phase fraction by flow cytometry and a faster doubling time of 24-36 h compared with 72 h for parental cells. The cyclin-dependent kinase inhibitor p27(kip1) was decreased in TR cells, and cyclin-dependent kinase 2 activity was increased. Importantly, exogenous addition of p27(kip1) increased trastuzumab sensitivity. Additionally, resistant cells displayed heightened sensitivity to the proteasome inhibitor MG132, which induced p27(kip1) expression. Thus, we propose that trastuzumab resistance may be associated with decreased p27(kip1) levels and may be susceptible to treatments that induce p27(kip1) expression.     

Neoplastic transformation of mammary epithelial cells in rats is associated with decreased apoptotic cell death

Previous studies have shown that terminal end buds (TEBs) in the murine mammary gland have high proliferative activity and demonstrate apoptotic cell death (ACD). Since TEBs are considered the place of origin of most chemically induced mammary carcinomas, we hypothesized that the development of hyperplastic and premalignant (carcinoma in situ, CIS) lesions in TEBs is associated with either a further increase in cell proliferation and/or with a decrease in ACD. To test this hypothesis we used the N-methyl-N-nitorosourea (MNU) carcinogenesis model in rats, where the occurrence of mammary tumors is preceded by hyperplastic and premalignant lesions arising mostly in TEBs, as well as in ducts and alveoli. The percentage of proliferating cells, as evaluated by 5-bromodeoxyuridine labeling (BrdU-LI), was similar in TEBs to those in terminal endbud hyperplasia (TEBH), CIS, and carcinomas (CA), whereas the percentage of apoptotic cells (apoptotic index, AI) was relatively high in TEBs and decreased in TEBH, CIS, and CA. This indicates that neoplastic transformation of mammary epithelial cells in TEBs is not associated with an increase in cell proliferation, but with a decrease in ACD. In addition to TEBH, hyperplastic lesions developed in ductal branching areas (ductal hyperplasia, DH) and alveolar structures (alveolar hyperplasia, AH). However, BrdU-LI in both DH and AH was lower than in TEBH, whereas the AI values were similar, suggesting that TEBH has a higher potential for progression and malignant transformation than DH and AH. In mammary tumors apoptotic cells were rare in the peripheral, proliferative areas, but frequent close to the necrotic areas, suggesting that intratumoral factors may significantly affect ACD. Thus, it appears that dissociation between cell proliferation and apoptosis occurs in the hyperplastic stages of mammary carcinogenesis and that neoplastic transformation of mammary epithelial cells is associated with decreased ACD but not with increased cell proliferation.     

The p53-dependent apoptotic pathway of breast cancer cells (BC-M1) induced by the bis-type bioreductive compound aziridinylnaphthoquinone

Introduction  

Several aziridinylbenzoquinone drugs have undergone clinical trials as potential antitumor drugs. These bioreductive compounds are designed to kill cells preferentially within the hypoxia tumor microenvironment. The bioreductive compound of bis-type naphthoquinone synthesized in our laboratory, 2-aziridin-1-yl-3-[(2-{2-[(3-aziridin-1-yl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio]ethoxy}ethyl)thio]naphthoquinone (AZ-1), had the most potent death effect on the breast cancer cells BC-M1 in our previous screening. In the present study, we determined that the mechanism of the death effect of BC-M1 cells induced by AZ-1 was mediated by the apoptosis pathway.     

Cisplatin (CDDP)-induced radiation resistance is not associated with CDDP resistance in 86HG39 and A172 malignant glioma cells

Malignant gliomas are often treated with cisplatin (cis-diamminedichloroplatinum(II), CDDP) and radiation but results remain unsatisfactory. The development of resistance might explain the poor prognosis. The aim of this study was to investigate whether two human malignant glioma cell lines, 86HG39 and A172, develop resistance to CDDP and/or radiation after CDDP pretreatment. The cells are incubated three times with 10⁻⁶ M CDDP for 24 h, allowed to recover from the treatment and then tested for sensitivity to CDDP and radiation (9 Gy, ⁶⁰Co) using a colorimetric assay (MTT). A172 pretreated and wild-type cells showed no difference in sensitivity to CDDP, whilst 86HG39 cells became more sensitive following pretreatment. This indicates that no resistant phenotype towards CDDP developed in any of the cell lines. In contrast, the CDDP-pretreated cells, after radiation, had significantly higher growth rates compared with the wild-type cells in both cell lines (A172: 4.4±0.5 versus 2.5±0.3, respectively, 192 h after radiation; 86HG39: 6.2±0.7 versus 2.3±0.3, respectively, 216 h after radiation; P<0.05) indicating resistance against radiation. The level of glutathione (GSH), which is known to mediate resistance against radiation, was 157.2±61.3 ng/mg protein in A172 cells and 93.2±16.9 ng/mg protein in 86HG39 cells, and there was no significant difference between levels in wild-type and pretreated cells (A172: 130.1±34; 86HG39: 81.6±10.4). These data show that CDDP pretreatment can induce resistance against radiation in vitro independently of CDDP cross-resistance mediated by a mechanism different from GSH.     

Cisplatin (platidiame) in the treatment of patients with disseminated breast cancer

In search of fresh indications for clinical application of cis-platinum, it was added to chemotherapy, the only treatment given to patients with disseminated breast cancer. The study group included 13 patients, with the data on 12 of them being subject to evaluation. The results showed a high antitumor effect of cis-platinum: objective remission was observed in 5 out of 12 cases (46.6%), with the complete 10 month-long remission included. All patients suffered from such untoward side-effects as sickness and vomiting. Moderate nephrotoxicity, which did not interfere with treatment, was registered in 3 cases. Since results confirmed the therapeutic effect of cis-platinum in breast cancer patients previously untreated with antitumor drugs further research into the problem should be recommended.     

The microtubule stabilizer patupilone (epothilone B) is a potent radiosensitizer in medulloblastoma cells

Concurrent radiochemotherapy for medulloblastoma includes the microtubule disrupting agent vincristine; however, vincristine alone or as part of a combined treatment regimen is highly toxic. A major goal is therefore to replace vincristine with novel potent chemotherapeutic agents-in particular, with microtubule stabilizing and destabilizing compounds-with a larger therapeutic window. Here, we investigated the antiproliferative, cytotoxic and radiosensitizing effect of patupilone (epothilone B [EPO906]), a novel, non-taxane-related and nonneurotoxic microtubule-stabilizing agent in human medulloblastoma cell lines. The antiproliferative and cytotoxic effects of patupilone alone and in combination with ionizing radiation was determined in the 3 representative human medulloblastoma cell lines D341Med, D425Med, and DAOY. Patupilone alone effectively reduced the proliferative activity and clonogenicity of all medulloblastoma cell lines tested at picomolar concentrations (50-200 pM) and resulted in an at least additive anticlonogenic effect in combination with clinically relevant doses of ionizing radiation (2 or 5 Gy). Cell-cycle analysis revealed a sequential G2-M arrest and sub-G1 accumulation in a dose- and treatment-dependent manner after exposure to patupilone. In tumor xenografts derived from D425Med cells, a minimal treatment regimen with patupilone and fractionated irradiation (1 × 2 mg/kg plus 3 × 3 Gy) resulted in an extended tumor growth delay for the 2 single treatment modalities alone and a supra-additive treatment response for the combined treatment modality, with complete tumor regressions. These results demonstrate the potent efficacy of patupilone against medulloblastoma cell lines and indicate that patupilone represents a promising candidate to replace vincristine as part of a combined treatment strategy with ionizing radiation.