Over-expression of APAF-1 and caspase-9 augments radiation-induced apoptosis in U-373MG glioma cells.

The p53 tumor-suppressor gene plays a critical role in radiation-induced apoptosis. Several genes, including Bax and Fas, are involved in p53-mediated apoptosis, and their over-expression enhances the degree of radiation-induced apoptosis. Apaf-1 and caspase-9 have been reported to be downstream components of p53-mediated apoptosis, suggesting that these genes play a role in radiation-induced apoptosis. In this study, we transduced U-373MG cells harboring mutant p53 with the Apaf-1 and/or caspase-9 genes via adenoviral (Adv) vectors concomitant with X-ray irradiation and evaluated the degree of apoptosis. The percentage of apoptotic cells in U-373MG cells co-infected with the Adv for Apaf-1 (Adv-APAF-1) and that for caspase-9 (Adv-Casp9) and treated with irradiation (24%) was much higher than that in cells co-infected with Adv-APAF-1 and Adv-Casp9 and not treated with irradiation (0.86%) and that in cells infected with either Adv-APAF-1 or Adv-Casp9 and treated with irradiation (2.0% or 2.6%, respectively). The apoptosis induced by co-transduction of Apaf-1 and caspase-9 and irradiation was repressed in cells that were co-infected with the Adv for Bcl-X(L) but not in cells co-infected with the Adv for Bcl-2. These results indicate that Apaf-1 and caspase-9 play a role in radiation-induced apoptosis in cancer cells harboring mutant p53. Bcl-X(L) may be critically involved in the radioresistance of cancer cells by repressing Apaf-1- and caspase-9-mediated apoptosis. Expression of Apaf-1 and caspase-9 in tumors may be an important determinant of the therapeutic effect of irradiation in cancer treatment.     

Co-transduction of p27Kip1 strongly augments Fas ligand- and caspase-8-mediated apoptosis in U-373MG glioma cells

BACKGROUND: p27Kip1 is a potential tumor suppressor gene. As malignant gliomas express Fas at high levels, the relationship between Fas-mediated apoptosis and p27Kip1 expression may improve therapeutic approaches for treating gliomas. MATERIALS AND METHODS: In this study, we transduced U-373MG glioma cells with the Fas ligand or caspase-8 genes using adenovirus vectors after transduction of the p27Kip1 gene to induce cell cycle arrest in U-373MG cells, and evaluated the degree of apoptosis. RESULTS: The results demonstrate that expression of p27Kip1 enhanced Fas ligand- or caspase-8-mediated apoptosis in U-373MG cells. Expression of apoptosis-related genes such as Bax, Bcl-X(L), Bcl-2 or caspase-8 were reduced by p27Kip1 transduction compared with that of beta-actin, whereas p27Kip1 transduction did not affect the expression level of Fas or the Fas ligand. CONCLUSION: Combined transduction of p27Kip1 with Fas ligand or caspase-8 would overide the resistance mechanism to apoptosis in malignant gliomas.     

Co-transduction of Apaf-1 and Caspase-9 Augments Etoposide-induced Apoptosis in U-373MG Glioma Cells

Several apoptosis-related genes have been reported to be involved in chemotherapy-induced apoptosis in cancers. An assessment of the relationship between expression of those genes and the degree of chemotherapy-induced apoptosis may be useful in improving the efficacy of cancer therapy. We transduced Apaf-1 (apoptotic protease-activating factor-1) and caspase-9 into U-373MG glioma cells using adenovirus (Adv) vectors in the presence of etoposide and evaluated the degree of apoptosis. The degree of apoptosis in etoposide-treated U-373MG cells infected with Adv for Apaf-1 (Adv-APAFl) was higher (27%) than that in cells infected with control Adv (14%), that in cells infected with Adv for caspase-9 (Adv-Casp9) was higher (34%) than that in cells infected with Adv-APAFl, and that in cells infected with both Adv-APAFl and Adv-Casp9 was the highest (41%). Treatment with etoposide increased expression of p53 and decreased expression of Bcl-X_L in U-373MG cells which harbored mutant p53. These results indicate that the expression of Apaf-1 and caspase-9 may be important determinants in predicting the sensitivity of cancers to chemotherapy. Adv-mediated co-transduction of Apaf-1 and caspase-9 should render cancer cells highly sensitive to chemotherapy.     

Adenovirus-mediated transfer of Fas ligand gene augments radiation-induced apoptosis in U-373MG glioma cells.

Most malignant astrocytomas (gliomas) express a high level of Fas, whereas the surrounding normal tissues such as neurons and astrocytes express a very low level of Fas. Thus, transduction of Fas ligand would selectively kill malignant astrocytoma cells. On the other hand, glioma cells harboring p53 mutation have been reported to be resistant to conventional therapies including radiation. To override the resistance mechanism of glioma cells with p53 mutation to radiation, we transduced U-373MG malignant astrocytoma (glioma) cells harboring mutant p53 with Fas ligand via an adenovirus (Adv) vector in combination with X-ray irradiation, and evaluated the degree of apoptosis. The degree of apoptosis in U-373MG cells infected with the Adv for Fas ligand (Adv-FL) and treated with irradiation (81%) was much higher than that in U-373MG cells infected with Adv-FL and not treated with irradiation (0.8%) or that in U-373MG cells infected with the control Adv for lacZ and treated with irradiation (5.0%). In U-373MG cells infected with Adv-FL, irradiation increased the expression of Fas ligand. Coincident with the increase in Fas ligand, there was a marked reduction in the caspase-3 level and a marked increase in the cleaved form of poly(ADP-ribose) polymerase (PARP), which are downstream components of Fas ligand-mediated apoptosis. This suggests that the enhanced activation of caspase-3 by the transduction of Fas ligand combined with irradiation, induced extensive apoptosis in U-373MG cells. In summary, transduction of Fas ligand may override the resistance mechanism to radiotherapy in glioma cells harboring p53 mutation.     

Co-transduction of Apaf-1 and caspase-9 highly enhances p53-mediated apoptosis in gliomas

Mutation of the p53 gene plays a critical role in the development of cancer and response to cancer therapy. To analyze the mechanism of cancer development and to improve cancer therapy, it is important to assess which genes are downstream components of p53 in cancers, and whether the expression levels of these genes affect p53-mediated apoptosis. In this study, we transduced the wild type p53 gene along with the Apaf-1 and caspase-9 genes via adenovirus vectors into U251 and U-373MG glioma cells harbouring a mutated p53, and evaluated the degree of apoptosis. Co-induction of Apaf-1 and caspase-9 genes highly enhanced p53-mediated apoptosis in glioma cells. Induction of wild type p53 enhanced the expression levels of Bax, p21/WAF1, and Fas protein. To determine which gene is activated by wild type p53 induction and, in turn, activates Apaf-1 and caspase-9, we transduced the Bax, p21/WAF1 or Fas gene via adenovirus vector to U251 cells to achieve a similar expression level as that induced by the Adv for p53 in U251 cells. U251 cells transduced with Fas concomitant with the Apaf-1 and caspase-9 genes underwent drastic apoptosis. This suggests that induction of wild type p53 upregulates Fas, which in turn may play a role in the activation of Apaf-1 and caspase-9. These results are important for analyzing the mechanism of tumour development and for predicting the therapeutic effect of p53 replacement gene therapy in a particular patient.     

Effects of amiloride treatment on U-118 MG and U-251 MG human glioma and HT-29 human colon carcinoma cells.

Human glioma (U-118 MG, U-251 MG) and human colon carcinoma (HT-29) spheroids and monolayers were continuously exposed to amiloride under physiological Na+ and HCO3- conditions. Amiloride in concentrations of 0.1-0.2 mM inhibited growth, while 0.5 mM or higher induced disintegration of the glioma spheroids within 4-6 days. Growth retardation of the HT-29 spheroids was achieved at concentrations of 0.4-0.5 mM and total growth inhibition and disintegration were achieved at 1.0 mM. Monolayer cultures of glioma cells were also more sensitive to amiloride than those of colon carcinoma cells. The higher amiloride concentrations induced pyknotic nuclei mainly in the central areas of the spheroids where the extracellular pH (pHe) was low. The amiloride-sensitive glioma spheroids had lower pHe than the colon carcinoma spheroids. The intracellular pH (pHi), measured in monolayers, was higher (7.11-7.18) in glioma cells than in colon carcinoma cells (6.94). High concentrations of amiloride, 1.0 mM for 1 h in combination with low Na+ concentrations, caused a strong pHi decrease in glioma cells but only a slight decrease in the colon carcinoma cells. The pHi measurements in glioma monolayers were carried out after 2-6 days of continuous exposure to 0.1 mM amiloride at physiological levels of Na+ and HCO3- to simulate the conditions during growth inhibition. After several days this caused, when growth already was inhibited, an acidification of pHi. Parallel measurements with X-ray microanalysis showed an increase of intracellular sodium and a decrease of intracellular potassium in the gliomas, while no such changes were seen in the colon carcinoma cells under identical conditions. It is concluded that the two glioma cell lines were more sensitive to amiloride, both as monolayers and spheroids, than the corresponding cultures of the colon carcinoma cell line. The inhibition of proliferation by amiloride seemed not to have a clear connection to pHi regulation.     

咖啡因通过FAK/AKT/ROCK通路调控人脑胶质瘤U-373MG细胞的恶 性生物学行为

目的：探讨咖啡因通过调节FAK/AKT/ROCK信号通路来影响人脑胶质瘤U-373MG细胞的增殖、迁移和侵袭能力。方法：常规培养U-373MG细胞,将其分为对照组、咖啡因低剂量（1 mmol/L）组、咖啡因高剂量（2 mmol/L）组、PF573228组（FAK抑制剂,1 μmol/L）、咖啡因高剂量+SC79组（AKT激活剂,8 mg/L）。用CCK-8法、Transwell小室实验、流式细胞术和WB法分别检测各组U-373MG细胞的增殖、迁移、侵袭及凋亡,以及U-373MG细胞中p-FAK、p-AKT、p-ROCK、Ki67、MMP-9蛋白表达水平。建立U-373MG细胞裸鼠移植瘤模型,观察咖啡因对移植瘤生长的影响,WB法检测移植瘤组织中相关蛋白的表达。结果：咖啡因、PF573228可显著抑制U-373MG细胞的增殖、迁移、侵袭能力,促进U-373MG细胞凋亡,抑制p-FAK、p-AKT、p-ROCK、Ki67、MMP-9蛋白的表达（均P<0.05）,SC79则可部分逆转咖啡因对U-373MG细胞的作用（均P<0.05）。咖啡因可显著抑制移植瘤的生长及移植瘤组织中上述相关蛋白的表达（均P<0.05）。结论：咖啡因可通过抑制FAK/AKT/ROCK信号通路抑制U-373MG细胞的增殖、迁移和侵袭能力,并促进其凋亡。     

The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis

Ectopic overexpression of Apaf-1 (2.5-fold) in human acute myelogenous leukemia HL-60 cells (HL-60/Apaf-1 cells) induced apoptosis and sensitized HL-60/Apaf-1 cells to etoposide- and paclitaxel-induced apoptosis (C. Perkins et al., Cancer Res., 58: 4561-4566, 1998). In this report, we demonstrate that in HL-60/Apaf-1 cells, the activity of caspase-9 and -3 induced by Apaf-1 overexpression was associated with a significant increase (5-fold) in the cytosolic accumulation of cytochrome c (cyt c), loss of mitochondrial membrane potential (deltapsim), and an increase in the reactive oxygen species. These were also associated with the processing of procaspase-8 and Bid (cytosolic, proapoptotic BH3 domain containing protein). Transient transfection of Apaf-1 into the Apaf-1-containing mouse embryogenic fibroblasts (MEFs; Apaf-1+/- MEFs) or Apaf-1-/- MEFs also induced the processing of procaspase-9 and procaspase-8, Bid cleavage, and apoptosis. These events were secondary to the activity of the downstream caspases induced by Apaf-1. This conclusion is supported by the observation that in HL-60/Apaf-1 cells, ectopic expression of dominant negative caspase-9, its inhibitory short isoform caspase-9b, or XIAP or treatment with the caspase inhibitor zVAD (50 microM) inhibited Apaf-1-induced caspase-8 and Bid cleavage, mitochondrial deltapsim, release of cyt c, and apoptosis. In contrast, a transient transfection of dominant negative caspase-8 or CrmA or exposure to caspase-8 inhibitor zIETD-fmk inhibited the processing of procaspase-8 and Bid but did not inhibit the cytosolic accumulation of cyt c in either the untreated HL-60/Apaf-1 cells or the etoposide-treated HL-60/Apaf-1 and HL-60/neo cells. These results indicate that Apaf-1 overexpression lowers the apoptotic threshold by activating caspase-9 and caspase-3. This triggers the mitochondrial deltapsim and cyt c release into the cytosol through a predominant mechanism other than cleavage of caspase-8 and/or Bid. This mechanism may involve a cytosolic mitochondrial permeability transition factor, which may be processed and activated by the downstream effector caspases, thereby completing an amplifying feedback loop, which triggers the mitochondrial events during apoptosis.     

Anti-tumour activity of tachykinin NK1 receptor antagonists on human glioma U373 MG xenograft

Astrocytes harbour functional receptors to many neurotransmitters, including substance P (SP), an undecapeptide belonging to the tachykinin family of peptide transmitters. SP activates malignant glial cells to induce cytokine release and proliferation, both responses being relevant for tumour progression. In tumours developed in nude mice transplanted subcutaneously (s.c.) to U373 MG human glioma cells, the presence of SP was observed at immunohistochemistry. Although the administration of exogenous SP did not significantly affect the size or development of U373 MG xenograft, a role of SP in supporting glioma progression in vivo was highlighted by the tumour growth inhibition induced by highly specific and selective human tachykinin NK1 receptor antagonists (MEN 11467 and MEN 11149). The anti-tumour activity of MEN 11467 was observed both with s.c. or intravenous treatments and was partially reverted by the concomitant administration of exogenous SP. Doxorubicin did not show any activity in controlling U373 MG growth in this in vivo model. A novel therapeutic approach to treat malignant gliomas with tachykinin NK1 receptor antagonists is suggested by these findings.     

RNA interference against SPARC promotes the growth of U-87MG human malignant glioma cells

Malignant glioma is a highly invasive brain tumor resistant to conventional therapies. Secreted protein acidic and rich in cysteine (SPARC) has been shown to facilitate glioma invasion. However, the effects of SPARC on cell growth have yet to be adequately elucidated. In this study, we constructed a plasmid expressing shRNA against SPARC, evaluated the effect of SPARCshRNA on SPARC expression and then assessed its effect on cell growth in U-87MG cells. Using plasmid-delivered shRNA, we effectively suppressed SPARC expression in U-87MG cells. Cell growth curves and colony formation assay suggested that the introduction of SPARCshRNA resulted in an increase of cell growth and colony formation. We also showed that knockdown of SPARC expression was capable of promoting the cell cycle progression from the G1 to S?phase. However, no difference was found in the level of apoptosis. A molecular analysis of signal mediators indicated that the inhibition of p-c-Raf?(Ser259) and accumulation of p-GSK-3β?(Ser9) and p-AKT?(Ser473) may be connected with the growth promotion by SPARC?shRNA. Our study may provide an insight into the biological function of SPARC in glioma.     

Adenovirus-mediated Transfer of Fas Ligand Gene Augments Radiation-induced Apoptosis in U-373MG Glioma Cells

Most malignant astrocytomas (gliomas) express a high level of Fas, whereas the surrounding normal tissues such as neurons and astrocytes express a very low level of Fas. Thus, transduction of Fas ligand would selectively kill malignant astrocytoma cells. On the other hand, glioma cells harboring p53 mutation have been reported to be resistant to conventional therapies including radiation. To override the resistance mechanism of glioma cells with p53 mutation to radiation, we transduced U-373MG malignant astrocytoma (glioma) cells harboring mutant p53 with Fas ligand via an adenovirus (Adv) vector in combination with X-ray irradiation, and evaluated the degree of apoptosis. The degree of apoptosis in U-373MG cells infected with the Adv for Fas ligand (Adv-FL) and treated with irradiation (81%) was much higher than that in U-373MG cells infected with Adv-FL and not treated with irradiation (0.8%) or that in U-373MG cells infected with the control Adv for lacZ and treated with irradiation (5.0%). In U-373MG cells infected with Adv-FL, irradiation increased the expression of Fas ligand. Coincident with the increase in Fas ligand, there was a marked reduction in the caspase-3 level and a marked increase in the cleaved form of poly(ADP-ribose) polymerase (PARP), which are downstream components of Fas ligand-mediated apoptosis. This suggests that the enhanced activation of caspase-3 by the transduction of Fas ligand combined with irradiation, induced extensive apoptosis in U-373MG cells. In summary, transduction of Fas ligand may override the resistance mechanism to radiotherapy in glioma cells harboring p53 mutation.     

Cyclooxygenase-2 inhibitor celecoxib augments chemotherapeutic drug-induced apoptosis by enhancing activation of caspase-3 and -9 in prostate cancer cells

Many tumors constitutively express high levels of the inducible form of proinflammatory enzyme, cyclooxygenase-2 (COX-2). Increased COX-2 expression is associated with tumor cell resistance to many cytotoxic chemotherapy drugs. Furthermore, increased resistance to cytotoxic antitumor drugs is also known to be dependent on associated stromal cells in many tumors. We investigated whether prostate tumor-associated stromal cells, marrow-derived osteoblasts, affect cytotoxicity of 2 antitumor drugs, COL-3 and docetaxel (TXTR), and whether it is dependent on COX-2 activity. We further examined whether inhibiting the activity of COX-2 negate the stroma-induced decrease in drug sensitivity in tumor cells. COX-2-specific inhibitor celecoxib (CXB) was used to inhibit COX-2 activity and associated alteration in cell death signaling was investigated. Coculturing PC-3ML cells with osteoblasts decreased the cytotoxicity of the tested antitumor drugs and was associated with increased COX-2 activity in PC-3ML cells. A significant decrease in drug-induced PGE(2) increase and an increase in cytotoxicity were observed when cells were treated with COL-3 or TXTR combined with CXB. Cytotoxicity of single or combination treatment increased apoptosis, which was associated with caspase-3 and -9 activation, PARP cleavage, increased BAD protein, but decreased protein levels of XIAP and BCL-(xL). Oral administration of CXB (40 mg/kg) to mice with PC-3ML tumors for 42 days increased tumor latency, decreased tumor growth and enhanced tumor control with COL-3 or TXTR. Overall, a synergistic enhancement of antitumor activity in combination treatment was observed in vitro and an additive effect in vivo. These observations suggest a potential clinical use of combined dosing of COX-2 inhibitors and cytotoxic drugs at lower, nontoxic dose than currently used to treat advanced prostate cancer.     

Adenovirus-mediated transfer of p53 augments hyperthermia-induced apoptosis in U251 glioma cells

PURPOSE: Hyperthermia kills glioma cells by inducing apoptosis and is thereby an effective therapeutic modality for the treatment of malignant gliomas. However, cells harboring mutated p53 are refractory to hyperthermia-induced apoptosis. In this study, we assessed whether or not adenovirus (Adv)-mediated transduction of p53 overrides this resistant mechanism. METHODS AND MATERIALS: We transduced the p53 wild-type tumor suppressor gene into U251 glioma cells harboring mutated p53 using Adv vectors in combination with hyperthermia (43, 44.5 degrees C), and evaluated the degree of cell death and apoptosis. RESULTS: The percentage of cells that had died, as measured by trypan blue staining, among U251 cells infected with the Adv for p53 (Adv-p53) and treated with hyperthermia, was significantly higher than the percentage of cells that had died among U251 cells infected with Adv-p53 and not treated with hyperthermia, or those infected with the control Adv for dE (Adv-dE) and treated with hyperthermia. The degree of apoptosis, measured at 24 h after treatment, in hyperthermia-treated U251 cells infected with Adv-p53 (43 degrees C, 73%; 44.5 degrees C, 92%) was much higher than that infected with Adv-p53 (41%), or that infected with control Adv-dE and treated with hyperthermia (43 degrees C, 1.3%; 44.5 degrees C, 19%). Treatment with combined hyperthermia and Adv-p53 infection induced cleavage of caspase-3 in U251 cells. CONCLUSION: These results indicate that Adv-mediated transduction of p53 would render glioma cells highly sensitive to hyperthermia.     

Histone deacetylase inhibitors, N-butyric acid and trichostatin A, induce caspase-8- but not caspase-9-dependent apoptosis in human malignant glioma cells

Histone deacetylase (HDAC) inhibitors have both apoptotic and differentiating effects on various tumor cells. However, the mechanisms underlying the effect of HDAC inhibitors remain unclear. In this study, we investigated the function of anti-proliferative effects of HDAC inhibitors, N-butyric acid and trichostatin A, on human malignant glioma cell lines, U251-MG and D54. MTT assay showed a dose-dependent inhibition of cellular proliferation in both cell lines. Cell cycle analysis revealed increased sub-G1 population in both lines, and G1 arrest only in U251-MG cells. Induction of apoptosis was also supported by the occurrence of DNA fragmentation in tumor cells treated with HDAC inhibitors. Furthermore, caspase inhibition assay indicated that HDAC inhibitor-induced apoptosis was caspase-dependent. Neither mitochondrial membrane potential nor the expression of caspase-9 was changed by treatment with HDAC inhibitors, suggesting the possibility that HDAC inhibitor-induced apoptosis was not mediated by the mitochondrial cell death pathway. On the other hand, immunoblot assay confirmed increased expression of caspase-8 in both lines, and elevation of p21 but not p27 protein in U251-MG cells following HDAC inhibitor treatment. Taken together, the HDAC inhibitors, N-butyric acid and trichostatin A, induce caspase-8- but not caspase-9-dependent apoptosis with or without p21-mediated G1 arrest in human malignant glioma cells.     

腺病毒介导IL-24基因诱导人脑胶质瘤U87MG细胞的凋亡

目的：研究腺病毒介导IL-24基因表达载体(Ad-IL-24)对人脑胶质瘤U87MG细胞的抑制作用,初步探讨其作用机制。方法：将本科室构建的Ad-IL-24感染U87MG细胞,RT-PCR法检测IL-24基因的表达,MTT法和流式细胞术检测U87MG细胞的生长和凋亡,激光共聚焦显微镜观察U87MG细胞凋亡的形态学变化,RT-PCR法检测Bax、Bcl-2基因mRNA的表达,Western blotting检测caspase-3的活化。结果：Ad-IL-24感染U87MG细胞后,IL-24在U87MG细胞中有明显表达,并抑制U87MG细胞生长、诱导其凋亡、出现典型的凋亡细胞核形态学改变。Ad-IL-24可上调U87MG细胞中Bax基因、下调Bcl-2基因的表达,并诱导caspase-3蛋白的活化。结论：Ad-IL-24可诱导U87MG细胞凋亡,其机制可能与上调Bax基因、下调Bcl-2基因表达,并活化caspase-3有关。     

Sequential two-step cleavage of the retinoblastoma protein by caspase-3/-7 during etoposide-induced apoptosis.

During cellular apoptosis, retinoblastoma protein (RB) is subjected to cleavage near the carboxyl terminus by a caspase-3-like protease. In addition, an heretofore unidentified protease cleaves RB internally, generating fragments of 68 and 48 kDa. Internal cleavage abrogates the ability of RB to associate with E2F. To investigate the mechanism of RB internal cleavage, we developed and employed an in vitro cleavage assay. Incubation of in vitro translated (35)S-RB with apoptotic cell extracts led to RB cleavage at the C-terminus, followed by internal cleavage. The caspase peptide inhibitors z-VAD-FMK or z-DEVD-FMK blocked both cleavage events. Rapid C-terminal and internal cleavage were also observed when recombinant caspase-3 was added to (35)S-RB. Moreover, when caspase-3 was added to nonapoptotic cell extract, efficient internal cleavage of cellular RB was observed. Caspase-mediated internal cleavage occurred following RB residue aspartate(349) in the sequence DSID(349). This sequence is consistent with a DXXD recognition motif for caspase-3-like enzymes. Interestingly, we also observed RB internal cleavage in caspase-3-deficient MCF-7 cells, indicating that other caspases are capable of cleaving RB internally. Indeed, caspase-7, a member of the caspase-3 subfamily, was found to cleave (35)S-RB at both the carboxyl terminus, and following aspartate(349). By contrast, caspases that are not members of the caspase-3 subfamily failed to cleave RB. Taken together, our findings demonstrate that during apoptosis, a caspase-3-like protease is responsible for degradation and functional inactivation of RB by cleaving the protein internally following aspartate(349).     

ATM and LKB1 dependent activation of AMPK sensitizes cancer cells to etoposide-induced apoptosis

The present study aims to determine the effect of AMPK on etoposide-induced apoptosis of cancer cells. Our results revealed that etoposide induced AMPK activation in prostate C4-2 cancer cells, an event that was attenuated by ATM siRNA. In A549 cells that lack LKB1, AMPK was unable to be activated by etoposide, which was restored by introduction of LKB1. Likewise, silencing LKB1 in C4-2 cells impaired AMPK activation. Finally, etoposide displayed a potent pro-apoptotic effect in cancer cells with functional LKB1 and AMPK. Thus, our results establish a linear relationship of ATM, LKB1 and AMPK in response to the DNA damage drug.