Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors

The mammalian target of rapamycin (mTOR) is a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and a central modulator of cell proliferation in malignant gliomas. Therefore, the targeting of mTOR signaling is considered a promising therapy for malignant gliomas. However, the mechanisms underlying the cytotoxic effects of a selective mTOR inhibitor, rapamycin, on malignant glioma cells are poorly understood. The purpose of this study was thus to elucidate how rapamycin exerts its cytotoxic effects on malignant glioma cells. We showed that rapamycin induced autophagy but not apoptosis in rapamycin-sensitive malignant glioma U87-MG and T98G cells by inhibiting the function of mTOR. In contrast, in rapamycin-resistant U373-MG cells, the inhibitory effect of rapamycin was minor, although the phosphorylation of p70S6 kinase, a molecule downstream of mTOR, was remarkably inhibited. Interestingly, a PI3K inhibitor, LY294002, and an Akt inhibitor, UCN-01 (7-hydroxystaurosporine), both synergistically sensitized U87-MG and T98G cells as well as U373-MG cells to rapamycin by stimulating the induction of autophagy. Enforced expression of active Akt in tumor cells suppressed the combined effects of LY294002 or UCN-01, whereas dominant-negative Akt expression was sufficient to increase the sensitivity of tumor cells to rapamycin. These results indicate that rapamycin exerts its antitumor effect on malignant glioma cells by inducing autophagy and suggest that in malignant glioma cells a disruption of the PI3K/Akt signaling pathway could greatly enhance the effectiveness of mTOR inhibitors.     

Synergistic interaction between 17-AAG and phosphatidylinositol 3-kinase inhibition in human malignant glioma cells

The phosphatidylinositol 3'-kinase (PI3K)/Akt pathway is often constitutively activated in malignant glioma cells, in many cases as a result of mutation of phosphatase and tensin homologue deleted on chromosome ten (PTEN), an endogenous inhibitor of Akt, which renders tumor cells resistant to cytotoxic insults, including those related to anticancer drugs. Pharmacological inhibition of this pathway may potentially restore or augment the effectiveness of conventional chemotherapy or other signaling-targeted agents. Because the heat shock protein (HSP) is involved in the conformational maturation of a number of signaling proteins critical to the proliferation of malignant glioma cells, we hypothesized that the combination of the PI3K inhibitor LY294002 and the HSP90 inhibitor 17-allyl-aminogeldanamycin (17-AAG) would promote glioma cytotoxicity by decreasing both the activation status and levels of Akt, as well as downregulating the levels of other relevant signaling effectors. We, therefore, examined the effects of LY294002 and 17-AAG, alone and in combination, on signal transduction and apoptosis in a series of malignant glioma cell lines. Simultaneous exposure to these inhibitors significantly induced cell death, and irreversibly inhibited proliferative activity and colony forming ability of the glioma cell lines. Quantitative analysis revealed that enhancement by LY294002 of 17-AAG-induced cytotoxicity was synergistic, leading to a pronounced increase in active caspase-3 and poly (adenosine diphosphate-ribose) polymerase (PARP) cleavage together with the release of cytochrome c and apoptosis inducing factor (AIF). No significant growth inhibition or caspase activation was seen in control cells. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and a significant downregulation of epidermal growth factor receptor (EGFR), Raf-1, and mitogen activated protein kinase. Combination of 17-AAG and LY294002 did not modify phospho-JNK/SPK and phospho-p38. Cells exposed to 17-AAG and LY294002 displayed a significant reduction in cell-cycle regulatory proteins, such as retinoblastoma (Rb), cyclin dependent kinase (CDK)4, CDK6, cyclin D1, and cyclin D3. Taken together, these findings suggest that the PI3K/Akt pathway plays a critical role in regulating the apoptotic response to 17-AAG and that targeting this pathway could provide a potent strategy to treat patients with malignant gliomas.     

The Cdk inhibitor flavopiridol enhances temozolomide-induced cytotoxicity in human glioma cells

The recent progress in chemotherapy for malignant gliomas is attributable to the introduction of the DNA-methylating agent temozolomide (TMZ); however, drug resistance remains a major issue. Previous studies have shown that TMZ induces prolonged arrest of human glioma cells in the G2/M phase of the cell cycle followed by a senescence-like phenomenon or mitotic catastrophe. These findings suggest that the G2 checkpoint is linked to DNA repair mechanisms. We investigated the effect of a cyclin-dependent kinase (Cdk) inhibitor flavopiridol (FP) that inhibits the action of Cdc2, a key protein in the G2 checkpoint pathway, on TMZ-treated glioma cells. Colony formation efficiency revealed that FP potentiated the cytotoxicity of TMZ in glioma cells in a p53-independent manner. This effect was clearly associated with the suppression of key proteins at the G2-M transition, accumulation of the cells exclusively at the G2 phase, and increase in a double-stranded DNA break marker (seen on performing immunoblotting). TMZ-resistant clones showed activation of the G2 checkpoint in response to TMZ, while FP treatment resensitized these clones to TMZ. FP also enhanced the cytotoxicity of TMZ in U87MG-AktER cells. Moreover, administration of TMZ and/or FP to nude mice with xenografted U87MG cells revealed that FP sensitized xenografted U87MG cells to TMZ in these mice. Our findings suggest that TMZ resistance could be promoted by enhanced DNA repair activity in the G2-M transition and that a Cdk inhibitor could suppress this activity, leading to potentiation of TMZ action on glioma cells.     

DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells

Although autophagy enhances cell survival in nutrient-deprived ceils by increasing adenosine triphosphate （ATP） production, it remains unclear if autophagy functions similarly in cells treated with cytotoxic chemotherapy agents. To address this issue, we measured both the ability of DNA damaging agents （Temozolomide, and Etoposide） to induce an autophagy-dependent production of ATP, and the effects of modulation of autophagy on drug-induced cell death.Both drugs induced an autophagy-associated increase in ATP production in multiple glioma cell lines. The drug-induced ATP surge could not be blocked by glucose starvation, but could be blocked by preincubation with the autophagy inhibitor 3-methyladenine （3-MA）, an siRNA targeting beclin 1, or the mitochondrial inhibitor oligomycin. Inhibition of autophagy-induced ATP production increased non-apoptotic cell death associated with micronucleation, while restoration of the 3-MA-inhibited ATP surge by addition of pyruvate suppressed cell death. These results show that DNA damaging agents induce an autophagy-associated ATP surge that protects cells and may contribute to drug resistance.     

Inhibition of phosphoinositide 3-kinase enhances the cytotoxicity of AG1478, an epidermal growth factor receptor inhibitor, in breast cancer cells

Aberrant activation and dysfunction of the EGFR/PI3K/Akt signaling pathways are commonly reported in breast cancer. Constitutive activation of the PI3K/Akt pathway by the lack of PTEN regulation is associated with resistance to novel targeted therapies including EGFR inhibitors. We aimed to study whether Ly294002, an inhibitor of PI3K, could enhance the cytotoxicity of AG1478, an inhibitor of EGFR, on breast cancer cells. We tested these agents in the MDA-MB-468 and MCF-7 breast cancer cell lines with different EGFR and PTEN profiles (MDA-MB-468: high expression of EGFR and PTEN mutation; MCF-7: low expression of EGFR and PTEN wild type). Simultaneous inhibition of EGFR and PI3K in MDA-MB-468 cells with combined Ly294002 and AG1478 treatment had a greater anti-proliferative effect and increased mitotic death than either treatment alone. In addition, more apoptosis and increased induction of cell arrest at G0/G1 phase were observed in MDA-MB-468 cells with the combined treatment. Phosphor-EGFR and its downstream signal transducer, phosphor-Akt, were fully attenuated only by simultaneous treatment with Ly294002 and AG1478. These data suggest that the inhibition of PI3K could enhance the cytotoxicity of EGFR inhibitors on breast cancer cells and tumors which overexpress EGFR and demonstrate mutated PTEN. This dual inhibition treatment protocol may have important therapeutic implication in the treatment of a subset of breast cancer patients with high expression of EGFR and deficient function of PTEN.     

Erythropoietin activates the phosphoinositide 3-kinase/Akt pathway in human melanoma cells

Erythropoietin (Epo) is used commonly to treat cancer and/or therapy-related anemia. Until recently, Epo was considered to be a specific stimulator of erythropoiesis, acting via its receptor, EpoR. It becomes clear, however, that EpoR is expressed in a variety of cell types other than hematopoietic cells, and that Epo is a potent cytoprotective cytokine increasing cell survival under hypoxic conditions. Epo and EpoR are also expressed in various malignant tumors, and EpoR expression shows association with tumor invasion and progression. Recently, a functional Epo autocrine signaling mechanism was also detected in human melanoma cells. In this study, we examined the hypothesis that Epo activates the Akt signaling pathway in human melanoma cells and thus promotes the survival of tumor cells. The Akt signaling pathway in response to Epo was examined in melanoma. Similar to Epo, the expression of EpoR was up-regulated in response to hypoxia and Epo stimulation in melanoma cells. Melanoma cells constitutively expressed Akt with variable expression of mammalian target of rapamycin, and Epo dose-dependently induced their activity. Epo increased Akt kinase activity, which was abrogated by co-treatment with LY294002, a specific blocker of phosphoinositide 3-kinase. LY294002 also inhibited the cytoprotective effects of Epo in melanoma cells under both normoxic and hypoxic conditions. Our results suggest that Epo promotes melanoma cell survival by activating an Akt-dependent signaling pathway.     

Inhibition of autophagy at a late stage enhances imatinib‐induced cytotoxicity in human malignant glioma cells

Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Imatinib mesylate, a tyrosine kinase inhibitor, is effective in the therapy of tumors including leukemias but not as a monotherapy for malignant glioma. Recently, it is thought that the adequate modulation of autophagy can enhance efficacy of anticancer therapy. The outcome of autophagy manipulation, however, seems to depend on the autophagy initiator, the combined stimuli, the extent of cellular damage and the type of cells, and it is not yet fully understood how we should modulate autophagy to augment efficacy of each anticancer therapy. In this study, we examined the effect of imatinib with or without different types of autophagy inhibitors on human malignant glioma cells. Imatinib inhibited the viability of U87‐MG and U373‐MG cells in a dose dependent manner and caused nonapoptotic autophagic cell death. Suppression of imatinib‐induced autophagy by 3‐methyladenine or small interfering RNA against Atg5, which inhibit autophagy at an early stage, attenuated the imatinib‐induced cytotoxicity. In contrast, inhibition of autophagy at a late stage by bafilomycin A1 or RTA 203 enhanced imatinib‐induced cytotoxicity through the induction of apoptosis following mitochondrial disruption. Our findings suggest that therapeutic efficiency of imatinib for malignant glioma may be augmented by inhibition of autophagy at a late stage, and that appropriate modulation of autophagy may sensitize tumor cells to anticancer therapy. © 2008 Wiley‐Liss, Inc.     

Suppression of tumor proliferation and angiogenesis of hepatocellular carcinoma by HS-104, a novel phosphoinositide 3-kinase inhibitor

Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway frequently instigates tumorigenesis leading to hepatocellular carcinoma (HCC). We synthesized N-(5-(3-(3-methyl-1,2,4-oxadiazol-3-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (HS-104), a novel PI3K inhibitor, and investigated its in vitro anticancer effect and in vivo capacity in an animal xenograft model. The inhibition of cell growth by HS-104 revealed that it was effective against HCC cell lines. Also, the activation of the AKT/mTOR signal cascade was inhibited by HS-104 treatment in a dose dependent manner. Flow cytometry analysis showed an accumulation of HCC cells in the G2/M phase with concomitant loss of cells in the S phase. The apoptotic effect of HS-104 was accompanied by increased evidence of cleaved caspase-3 and PARP, as well as DNA fragmentation. In angiogenesis studies, HS-104 inhibited the tube formation of vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs), and suppressed microvessel sprouting from a rat aortic ring, ex vivo, and blood vessel formation in the Matrigel plug assay in mice. HS-104 inhibited the expression of the downstream proteins of PI3K including p-AKT, p-mTOR and p-p70S6K in VEGF-induced HUVECs. In the xenograft animal model, HS-104 significantly delayed tumor growth in a dose dependent manner and suppressed the expression of PCNA, CD34 and cleaved caspase-3 in tumor tissue. These studies show that HS-104 inhibited the PI3K/AKT/mTOR signaling pathway resulting in cell growth/angiogenesis inhibition and apoptosis induction. Therefore, HS-104 is considered as a novel drug candidate for the treatment of HCC.     

Anticonvulsant drugs fail to modulate chemotherapy-induced cytotoxicity and growth inhibition of human malignant glioma cells

Adjuvant chemotherapy after cytoreductive surgery and irradiation plays an increasingly important role in the management of human malignant glioma. Here we have examined the effect of three anticonvulsants most commonly administered to glioma patients, carbamazepine, phenytoin and valproic acid, on the cytotoxic and antiproliferative actions in vitro of several cancer chemotherapy drugs currently evaluated for human gliomas. We find that none of the anticonvulsants reduces glioma cell viability or proliferation or modulates glioma cell clonogenicity at clinically relevant concentrations when administered alone. Therapeutic concentrations of either drug fail to alter the effect of cancer chemotherapy drugs in acute cytotoxicity assays or modified clonogenicity assays. A lack of interactions of anticonvulsants and cytotoxic drugs is also observed when the glioma cells are preexposed to the anticonvulsants for prolonged times, suggesting that chronic exposure to anticonvulsants in vivo may not change intrinsic glioma cell sensitivity to cancer chemotherapy. Thus, changes in hepatic enzyme activity or immunological parameters, but not modulation of intrinsic chemotherapeutic drug sensitivity, may influence the choice of an anticonvulsant for seizure control in glioma patients receiving adjuvant chemotherapy.     

Arsenic disulfide synergizes with the phosphoinositide 3-kinase inhibitor PI-103 to eradicate acute myeloid leukemia stem cells by inducing differentiation

Although dramatic clinical success has been achieved in acute promyelocytic leukemia (APL), the success of differentiating agents has not been reproduced in non-APL leukemia. A key barrier to the clinical success of arsenic is that it is not potent enough to achieve a clinical benefit at physiologically tolerable concentrations by targeting the leukemia cell differentiation pathway alone. We explored a novel combination approach to enhance the eradication of leukemia stem cells (LSCs) by arsenic in non-APL leukemia. In the present study, phosphatidylinositol 3-kinase /AKT/mammalian target of rapamycin (mTOR) phosphorylation was strengthened after As(2)S(2) exposure in leukemia cell lines and stem/progenitor cells, but not in cord blood mononuclear cells (CBMCs). propidium iodide-103, the dual PI3K/mTOR inhibitor, effectively inhibited the transient activation of the PI3K/AKT/mTOR pathway by As(2)S(2). The synergistic killing and differentiation induction effects on non-APL leukemia cells were examined both in vitro and in vivo. Eradication of non-APL LSCs was determined using the nonobese diabetic/severe combined immunodeficiency mouse model. We found that a combined As(2)S(2)/PI-103 treatment synergized strongly to kill non-APL leukemia cells and promote their differentiation in vitro. Furthermore, the combined As(2)S(2)/PI-103 treatment effectively reduced leukemia cell repopulation and eradicated non-APL LSCs partially via induction of differentiation while sparing normal hematopoietic stem cells. Taken together, these findings suggest that induction of the PI3K/AKT/mTOR pathway could provide a protective response to offset the antitumor efficacy of As(2)S(2). Targeting the PI3K/AKT/mTOR pathway in combination with As(2)S(2) could be exploited as a novel strategy to enhance the differentiation and killing of non-APL LSCs.     

X-irradiation to human malignant glioma cells enhances the cytotoxicity of autologous killer lymphocytes under specific conditions

PURPOSE: To clarify the effect of combining x-irradiation and human killer lymphocytes against autologous malignant glioma cells, we analyzed not only the alteration of surface antigen expression in irradiated tumor cells, but also the cytotoxic effects of human killer lymphocytes on the autologous tumor cells with and without x-irradiation. METHODS AND MATERIALS: Six malignant glioma cell-lines (MG 1-6) established from each patient with a malignant glioma in our institute, and U87MG, were used as materials. They were irradiated by 0-50 Gy of X-rays, and the alternations of their human histocompatability leukocyte antigen (HLA), HLA-ABC, HLA-DR, -DP, -DQ, and FAS expressions were examined. Then, three sets of autologous natural killer (NK) cells, and autologous tumor-specific T lymphocytes (ATTL) were induced from the peripheral blood mononuclear cells (PBMCs) of three patients, and in vitro cytotoxic effects of these killer cells on the irradiated autologous tumor cells were analyzed. RESULTS: Irradiation-enhanced HLA-DR, -DP, -DQ, and FAS expression in glioma cell lines with low p53 expression. However, there was no correlation between HLA-ABC expression and X-ray dose. After irradiation of the tumor cells, cytotoxicity was enhanced in four of six effectors; in particular, it was significantly elevated in two killer lymphocytes. It was speculated that the enhancing effect was influenced not only by the p53 status of the tumor, but also by the types of killer lymphocytes; the alteration of cytotoxicity in NK cells on irradiated tumor cells may be compensatory for alteration in ATTLs. CONCLUSION: It was indicated that irradiation of malignant tumor cells enhanced killer cell-mediated cytotoxicity in autologous models under specific conditions. These basic data should contribute to clinical trials using local radiotherapy and systemic adoptive immunotherapy with killer lymphocytes.     

Cytokine activation of phosphoinositide 3-kinase sensitizes hematopoietic cells to cisplatin-induced death

Cytokine growth factors regulate the normal proliferation of hematopoietic cells but can also override irradiation-induced growth arrest checkpoints through activation of a phosphoinositide 3-kinase (PI3K) signaling pathway. In the present study, we assessed the effect that erythropoietin and interleukin-3 have on cisplatin-treated hematopoietic cells. When cultured in the presence of cytokine, cisplatin-treated 32D cells transiently accumulated in a G(2)-M phase arrest and ultimately died by a nonapoptotic mechanism. By comparison, reduction of cytokine-induced PI3K activity, either through cytokine receptor mutation or direct inhibition with LY294002, caused cisplatin-treated cells to enter a biphasic G(1) and G(2)-M arrest. The arrest of these cells coincided with an absence of cyclin-dependent kinase (Cdk)1 and Cdk2 activity and significantly reduced cell death during cisplatin treatment. Indeed, LY294002 treatment during cisplatin exposure allowed the recovery of a viable, proliferating cell population after removal of cisplatin. In contrast, Cdks remained active in the G(2)-M-arrested population of cisplatin-treated cells with continuous cytokine activation of PI3K, and even transient exposure to cisplatin resulted in death of the entire population. These data suggest that cytokine activation of PI3K signaling pathways overrides cisplatin-induced growth arrest checkpoints, thereby sensitizing hematopoietic cells to DNA damage-induced death.     

Insulin receptor substrate is a mediator of phosphoinositide 3-kinase activation in quiescent pancreatic cancer cells

Phosphoinositide 3-kinase (PI3K) is activated in pancreatic cancer cells and plays a central role in their proliferation, survival, and drug resistance. Although the mechanism is unclear, PI3K activation in these cells could be due to physical interaction between its regulatory subunit (p85) and specific tyrosine kinases or their mediators. Consistent with this possibility, PI3K was precipitated with anti-phosphotyrosine antibodies and Akt phosphorylation was blocked by the tyrosine kinase inhibitors SU6656 and PD158780 in quiescent pancreatic cancer cells. Pull-down assays with a fusion protein (GST-p85NC-SH2), and coimmunoprecipitation studies, indicated that the insulin receptor substrate (IRS), and not the epidermal growth factor and insulin-like growth factor receptors or the Src tyrosine kinase, was physically associated with PI3K in these cells. Our data also indicated that SU6656 and PD158780 inhibited Akt activation in pancreatic cancer cells by interfering with the ability of IRS-1 to recruit PI3K. Furthermore, IRS-1 was phosphorylated on a p85-binding site (Y(612)), and IRS-specific small interfering RNA potently inhibited activation of PI3K and Akt in transfected cells. Taken together, these observations indicate that IRS is a mediator of PI3K activation in quiescent pancreatic cancer cells.     

Akt inhibitor shows anticancer and radiosensitizing effects in malignant glioma cells by inducing autophagy

Autophagy, or programmed cell death type II, is one of the responses of cancer cells to various therapies, including ionizing radiation. Recently, we have shown that radiation induces autophagy, but not apoptosis, in various malignant glioma cell lines. Autophagy is mainly regulated by the mammalian target of rapamycin (mTOR) pathway. The Akt/mTOR pathway also mediates oncogenesis and radioresistance. Thus, we hypothesized that inhibiting this pathway has both an anticancer and radiosensitizing effect by activating autophagy. The purpose of our study was therefore to determine whether and by which mechanisms an Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate, had anticancer and radiosensitizing effects on malignant glioma U87-MG and radioresistant U87-MG cells with a consistitutively active form of epidermal growth factor receptor (U87-MGDeltaEGFR). Treatment with the Akt inhibitor successfully inhibited Akt activity and reduced cell viability in both cell lines. In terms of the mechanism, the Akt inhibitor decreased phosphorylated p70S6 kinase, a downstream target of Akt, and induced autophagy, but not apoptosis. Furthermore, the Akt inhibitor radiosensitized both U87-MG and U87-MGDeltaEGFR cells by enhancing autophagy. Specific inhibition of Akt using the dominant-negative Akt plasmid also resulted in enhanced radiation-induced autophagy. In conclusion, an Akt inhibitor showed anticancer and radiosensitizing effect on U87-MG and U87-MGDeltaEGFR cells by inducing autophagy. Thus, Akt inhibitors may represent a promising new therapy as a single treatment or used in combination with radiation for malignant gliomas, including radioresistant ones that express DeltaEGFR.     

Quantitative cloning of malignant human glioma cells by conditioned medium.

The use of conditioned medium (CM) obtained from monolayer cultures of human glioma cells induced colony formation from single glioma cells in culture. In contrast, no colonies were observed in cultures incubated with nonconditioned standard Eagle's basal medium. The number of colonies formed closely depended on the concentration of CM. The glioma CM not only stimulated colony formation but also induced the formation of fibrillary cell extensions. Culture conditions influencing the production of colony-stimulating factors included cell density and duration of culture medium contact with glioma cells. The colony-stimulating activity (CSA) was stable after freezing and thawing, but decreased 30-40% when CM was exposed to temperatures over 66 degrees C for 30 minutes. In addition, the CSA was filtratable (0.45 mu), dialyzable, and passable through an Amicon PM10 filter, which indicated a molecular weight less than 10,000. The use of CM provided an improved method for quantitative assays of neural tumor cells, based on their colony formation in culture.     

Anticancer activity of HS-527, a novel inhibitor targeting PI3-kinase in human pancreatic cancer cells

Pancreatic cancer is known to have low 5-year survival rate and poor response to treatment. In this study, we synthesized HS-527, a new PI3-kinase inhibitor, and investigated not only its anticancer activity, but also its mechanism of action in pancreatic cancer cells. HS-527 had higher specificity for PI3K than other kinases and inhibited PI3K/Akt signaling pathway by down-regulating Akt and P70S6K. And HS-527 inhibited the cell growth and proliferation of the pancreatic cancer in a time- and dose-dependent manner, with greater activity than gemcitabine. Even HS-527 showed lower cytotoxicity than gemcitabine in normal cells. When treated with HS-527, the cancer cells appeared apoptotic, increasing the expression of cleaved PARP, cleaved caspase-3, and Bax. Furthermore, HS-527 showed an anti-angiogenic activity by decreasing the expression of HIF-1α and VEGF, and inhibited the migration of endothelial cells, and the formation of new blood vessel in mouse Matrigel plug assay. In this study, we found that HS-527 showed anti-cancer activity through an inhibition of the PI3K/Akt pathway in pancreatic cancer cells, suggesting that HS-527 could be used as a promising therapeutic agent for pancreatic cancer.     

Synergistic cytotoxicity of Bcl-xL inhibitor, gossypol and chemotherapeutic agents in non-Hodgkin's lymphoma cells

Anti-apoptotic proteins Bcl-2 and Bcl-xL are overexpressed in 80% of non Hodgkin's lymphoma cells and are thought to play an important role in the resistance of lymphoma cells to current chemotherapeutic agents. Gossypol, an orally-active polyphenolic aldehyde derived from the cotton plant, has been known to have potential anti-neoplastic activity. Recently, gossypol was found to bind to the BH3 binding groove of Bcl-xL and with lesser affinity to Bcl-2. The present study was conducted to determine whether gossypol increases the sensitivity of non-Hodgkin's lymphoma cells to the actions of chemotherapeutic agents by potentiating treatment-induced apoptosis. The interactions observed between gossypol and chemotherapeutic drugs were analyzed using the median effect principle (CalcuSyn analysis). Our data showed that treatment of Ramos cells with gossypol not only induced cell arrest on the G(0)/G(1) phase, but also augmented apoptosis and growth inhibition induced by etoposide (VP-16), doxorubicin hydrochloride (ADM), vincristine (VCR), and paclitaxel (taxol). However, when gossypol was combined with cisplatin (DDP) an antagonistic effect was observed. Gossypol-induced cell cycle arrest was accompanied by decreased expression of cyclin D1 in Ramos cells. In addition, the peroxisome proliferator-activated receptor (PARP) pathway is, at least in part, involved in the gossypol-induced apoptosis when combined with VP-16. These data indicate that single-agent gossypol is effective in inhibiting growth of non-Hodgkin's lymphoma cells in vitro and combination studies with certain secondary chemotherapeutic agents further demonstrate it's synergistic cytotoxicity. These findings support future preclinical and clinical studies of gossypol in the treatment of non-Hodgkin's lymphoma.     

Modulation of growth and radiochemosensitivity of human malignant glioma cells by acidosis

BACKGROUND: Glioblastoma commonly is characterized by hypoxia and acidosis and the histologic features of tissue necrosis and neovascularization. Current approaches of adjuvant radiochemotherapy for patients with glioblastoma have only a modest impact on the natural course of this disease. METHODS: The authors examined the effects of acidosis on growth and response to irradiation and chemotherapy in cultured human malignant glioma cells. RESULTS: The authors found that mild acidosis (pH 7.0) inhibited the growth of cell lines that retained wild type p53 activity but did not inhibit the growth of cell lines that were devoid of p53 function. Transfer of a dominant-negative p53 gene into p53 wild type cells failed to override the acidosis-conferred growth arrest, suggesting that loss of p53 activity per se does not mediate escape from acidosis-induced growth inhibition. Moderate acidosis (pH 6.6) inhibited the growth of all cell lines. Acidosis-mediated growth arrest was not associated with a specific type of cell cycle arrest, e.g., in G0/G1 or G2/M phase. Acidosis did not result in consistent changes in radiosensitivity; however, it enhanced the cytotoxic effects of lomustine but conferred protection from topotecan, vincristine, teniposide, and cisplatin cytotoxicity. Lomustine exhibited enhanced stability at low pH, providing a putative mechanism for the enhanced cytotoxic effects of lomustine in acidotic conditions. Decreased sensitivity to the other drugs did not result from altered multidrug resistance drug transport activity. CONCLUSIONS: Taken together, the current results suggest that tissue acidosis may be an important determinant of glioma cell responses to adjuvant radiochemotherapy. The superior activity of nitrosoureas, such as lomustine, compared with other agents in patients with glioblastoma may result in part from prolonged drug stability in an acidotic microenvironment.