Inhibition of autophagy enhances the effects of E1A-defective oncolytic adenovirus dl922-947 against glioma cells in vitro and in vivo

Oncolytic viruses represent a novel therapeutic approach for aggressive tumors, such as glioblastoma multiforme, which are resistant to available treatments. Autophagy has been observed in cells infected with oncolytic viruses; however, its role in cell death/survival is unclear. To elucidate the potential therapeutic use of autophagy modulators in association with viral therapy, we analyzed autophagy induction in human glioma cell lines U373MG and U87MG infected with the oncolytic adenovirus dl922-947. dl922-947 infection triggered an autophagic cellular response, as shown by the development of acidic vesicular organelles, LC3-I→LC3-II conversion, and reduction of p62 levels. However, on infection, the Akt/mTOR/p70s6k pathway, which negatively regulates autophagy, was activated, whereas the ERK1/2 pathway, a positive regulator of autophagy, was inhibited. Accordingly, MEK inhibition by PD98059 sensitized glioma cells to dl922-947 effects, whereas autophagy induction by rapamycin protected cells from dl922-947-induced death. Treatment with two inhibitors of autophagy, chloroquine and 3-methyladenine, increased the cytotoxic effects of dl922-947 in vitro. In vivo, the growth of U87MG-induced xenografts was further reduced by adding chloroquine to the dl922-947 treatment. In conclusion, autophagy acts as a survival response in glioma cells infected with dl922-947, thus suggesting autophagy inhibitors as adjuvant/neoadjuvant drugs in oncolytic virus-based treatments.     

The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human ovarian cancer cells

The role of autophagy in oncogenesis remains ambiguous, and mechanisms that induce autophagy and regulate its outcome in human cancers are poorly understood. The maternally imprinted Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI; also known as DIRAS3) is downregulated in more than 60% of ovarian cancers, and here we show that re-expression of ARHI in multiple human ovarian cancer cell lines induces autophagy by blocking PI3K signaling and inhibiting mammalian target of rapamycin (mTOR), upregulating ATG4, and colocalizing with cleaved microtubule-associated protein light chain 3 (LC3) in autophagosomes. Furthermore, ARHI is required for spontaneous and rapamycin-induced autophagy in normal and malignant cells. Although ARHI re-expression led to autophagic cell death when SKOv3 ovarian cancer cells were grown in culture, it enabled the cells to remain dormant when they were grown in mice as xenografts. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. However, inhibition of ARHI-induced autophagy with chloroquine dramatically reduced regrowth of xenografted tumors upon reduction of ARHI levels, suggesting that autophagy contributed to the survival of dormant cells. Further analysis revealed that autophagic cell death was reduced when cultured human ovarian cancer cells in which ARHI had been re-expressed were treated with growth factors (IGF-1, M-CSF), angiogenic factors (VEGF, IL-8), and matrix proteins found in xenografts. Thus, ARHI can induce autophagic cell death, but can also promote tumor dormancy in the presence of factors that promote survival in the cancer microenvironment.     

Trastuzumab signaling in ErbB2-overexpressing inflammatory breast cancer correlates with X-linked inhibitor of apoptosis protein expression

Inflammatory breast cancer (IBC) patients show poor survival and a significant incidence of epidermal growth factor receptor-2 (ErbB2) overexpression. A distinct mechanism involving increased expression of X-linked inhibitor of apoptosis protein (XIAP) and survivin, key members of the inhibitor of apoptosis protein (IAP) family, was observed post-trastuzumab (an ErbB2 monoclonal antibody) treatment in an ErbB2-overexpressing, estrogen receptor negative, IBC cellular model, SUM190PT, isolated from a primary IBC tumor. In contrast, a decrease in the IAP expression was observed in the non-IBC, ErbB2-overexpressing SKBR3 cells in which trastuzumab treatment also decreased p-AKT and cell viability. Further, in SUM190PT cells, therapeutic sensitivity to GW583340 (a dual epidermal growth factor receptor/ErbB2 kinase inhibitor) corresponded with XIAP down-regulation and abrogation of XIAP inhibition on active caspase-9 release. Specific small interfering RNA-mediated XIAP inhibition in combination with trastuzumab caused decrease in inactive procaspase-9 and inhibition of p-AKT corresponding with 45% to 50% decrease in cell viability in the SUM190PT cells, which have high steady-state p-AKT levels. Further, embelin, a small-molecule inhibitor that abrogates binding of XIAP to procaspase-9, caused significant decrease in SUM190PT viability. However, embelin in combination with trastuzumab failed to affect SUM190PT viability because it has no direct effect on XIAP, which is induced by trastuzumab treatment. These data have identified a novel functional link between ErbB2 signaling and antiapoptotic pathway mediated by XIAP. Blockade of the IAP antiapoptotic pathway alone or in combination would be an attractive strategy in IBC therapy.     

Induction of cytosine arabinoside-resistant human myeloid leukemia cell death through autophagy regulation by hydroxychloroquine

We investigated the effects of the autophagy inhibitor hydroxychloroquine (HCQ) on cell death of cytosine arabinoside (Ara-C)-resistant human acute myeloid leukemia (AML) cells. Ara-C-sensitive (U937, AML-2) and Ara-C-resistant (U937/AR, AML-2/AR) human AML cell lines were used to evaluate HCQ-regulated cytotoxicity, autophagy, and apoptosis as well as effects on cell death-related signaling pathways. We found that HCQ-induced dose- and time-dependent cell death in Ara-C-resistant cells compared to Ara-C-sensitive cell lines. The extent of cell death and features of HCQ-induced autophagic markers including increase in microtubule-associated protein light chain 3 (LC3) I conversion to LC3-II, beclin-1, ATG5, as well as green fluorescent protein-LC3 positive puncta and autophagosome were remarkably greater in U937/AR cells. Also, p62/SQSTM1 was increased in response to HCQ. p62/SQSTM1 protein interacts with both LC3-II and ubiquitin protein and is degraded in autophagosomes. Therefore, a reduction of p62/SQSTM1 indicates increased autophagic degradation, whereas an increase of p62/SQSTM1 by HCQ indicates inhibited autophagic degradation. Knock down of p62/SQSTM1 using siRNA were prevented the HCQ-induced LC3-II protein level as well as significantly reduced the HCQ-induced cell death in U937/AR cells. Also, apoptotic cell death and caspase activation in U937/AR cells were increased by HCQ, provided evidence that HCQ-induced autophagy blockade. Taken together, our data show that HCQ-induced apoptotic cell death in Ara-C-resistant AML cells through autophagy regulation.     

IL-17A and IL-17F induce autophagy in RAW 264.7 macrophages

Autophagy is an important cellular catabolic process for the lysosomal degradation of cytoplasmic organelles, proteins and microorganisms. The autophagic process is intertwined with the immune response: autophagy regulates both innate and adaptive immunity, conversely, cytokines produced during the course of the immune response modulate various functions of the autophagic cascade. The IL-17 family member cytokines play a pivotal role in immune protection against extra- and intracellular bacterial pathogens. Since the effects of IL-17A and IL-17F on autophagy have not yet been reported, we have evaluated the autophagic activity in the RAW 264.7 cell line treated with either IL-17A or IL-17F. Both IL-17A and IL-17F proved to promote microtubule-associated protein 1 light chain 3 B-II (LC3B-II) accumulation, enhance the autophagic flux, facilitate the intracellular redistribution of LC3B, increase both the average number and the size of autophagosomes per cell, and foster the formation of acidic vesicular organelles. IL-17F was considerably more efficient than IL-17A in promoting the autophagic process. Further experiments to determine the potential effect of IL-17-induced autophagy on the antibacterial activity of RAW macrophages revealed that IL-17F significantly decreased the intracellular counts of Mycobacterium terrae, while the colony-forming unit values remained comparable in the IL-17A-treated cells and the control cultures. In conclusion, these results demonstrate that IL-17A and IL-17F are capable of inducing autophagy in macrophages, and thereby contribute to the elimination of Mycobacterium terrae. These data may bear on the pathogenesis of infections caused by Mycobacterium terrae, as IL-17 plays a pivotal role in the immune response to mycobacteria. IL-17-mediated activation of autophagy may also be implicated in various infections and other pathological conditions.     

The endoplasmic reticulum stress induced by tunicamycin affects the viability and autophagy activity of chondrocytes

Osteoarthritis (OA) is attributed to a reduction in chondrocytes within joint cartilage, and research has shown that endoplasmic reticulum (ER) stress and autophagy play important roles in the survival of chondrocytes. However, the relationship between ER stress and autophagy in chondrocytes remains unclear. In this study, we investigated the changes in apoptotic and autophagic activity in chondrocytes under ER stress. Following treatment with tunicamycin, the rate of apoptosis among chondrocytes increased. Western blot analysis showed the levels of unfolded protein response (UPR) related proteins increased, followed by elevated expression of light chain 3B-II (LC3B-II) and Beclin-1. An ultrastructural investigation showed that a large number of pre‑autophagosomal structures or autophagosomes formed under tunicamycin treatment. However, the autophagy activity was significantly inhibited in chondrocytes after suppression of GRP78 by siRNA. The apoptosis ratio of chondrocytes pre-treated with 3-methyladenine was much higher than that of normal chondrocytes after exposure to tunicamycin. Our study revealed that the tunicamycin-induced persistent UPR expression led to apoptosis of chondrocytes and activation of autophagy incorporation with GRP78. Blocking autophagy accelerated the apoptosis induced by ER stress, which confirmed the protective function of autophagy in the homeostasis of chondrocytes. These findings advance our understanding of chondrocyte apoptosis and provide potential molecular targets for preventing apoptotic death of chondrocytes.     

Chloroquine Has a Cytotoxic Effect on Acanthamoeba Encystation through Modulation of Autophagy

Encystation of Acanthamoeba castellanii is associated with resistance to chemotherapeutic agents. Blocking the encystation process could potentiate the efficacy of chemotherapeutic agents and biocides. During encystation, autophagy is highly stimulated and required for proper encystation of Acanthamoeba. In this study, the cytotoxic effect of chloroquine, a well-known autophagy-inhibitory drug, was tested in A. castellanii. Chloroquine was able to selectively reduce cell survival during the encystation of A. castellanii. However, A. castellanii trophozoites and mature cysts were resistant to chloroquine. Chloroquine treatment led to an increase in the number and size of lysosomes in encysting cells. Moreover, chloroquine inhibited the degradation of long-lived proteins in the encysting cells. Decreased autophagic flux, indicated by an increased number of lysosomes and decreased degradation of long-lived proteins, may be the mechanism by which cell death is induced by chloroquine in encysting Acanthamoeba. These results suggest a potential novel therapeutic application of chloroquine as an anti-Acanthamoeba drug. Our findings also suggest that targeting autophagy could be a therapeutic strategy against Acanthamoeba infection.     

Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma

Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. Whether autophagy observed in treated cancer cells represents a mechanism that allows tumor cells to survive therapy or a mechanism for initiating a nonapoptotic form of programmed cell death remains controversial. To address this issue, the role of autophagy in a Myc-induced model of lymphoma generated from cells derived from p53ER(TAM)/p53ER(TAM) mice (with ER denoting estrogen receptor) was examined. Such tumors are resistant to apoptosis due to a lack of nuclear p53. Systemic administration of tamoxifen led to p53 activation and tumor regression followed by tumor recurrence. Activation of p53 was associated with the rapid appearance of apoptotic cells and the induction of autophagy in surviving cells. Inhibition of autophagy with either chloroquine or ATG5 short hairpin RNA (shRNA) enhanced the ability of either p53 activation or alkylating drug therapy to induce tumor cell death. These studies provide evidence that autophagy serves as a survival pathway in tumor cells treated with apoptosis activators and a rationale for the use of autophagy inhibitors such as chloroquine in combination with therapies designed to induce apoptosis in human cancers.     

Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab

Autophagy has been emerging as a novel cytoprotective mechanism to increase tumor cell survival under conditions of metabolic stress and hypoxia as well as to escape chemotherapy-induced cell death. To elucidate whether autophagy might also protect cancer cells from the growth inhibitory effects of targeted therapies, we evaluated the autophagic status of preclinical breast cancer models exhibiting auto-acquired resistance to the anti-HER2 monoclonal antibody trastuzumab (Tzb). We first examined the basal autophagic levels in Tzb-naive SKBR3 cells and in two pools of Tzb-conditioned SKBR3 cells (TzbR), which optimally grow in the presence of Tzb doses as high as 200 µg/ml Tzb. Fluorescence microscopic analyses revealed that the number of punctate LC3 structures -a hallmark of autophagy- was drastically higher in Tzb-refractory cells than in Tzb-sensitive SKBR3 parental cells. Immunoblotting analyses confirmed that the lipidation product of the autophagic conversion of LC3 was accumulated to high levels in TzbR cells. High levels of the LC3 lipidated form in Tzb-refractory cells were accompanied by decreased p62/sequestosome-1 protein expression, a phenomenon characterizing the occurrence of increased autophagic flux. Moreover, increased autophagy was actively used to survive Tzb therapy as TzbR pools were exquisitely sensitive to chemical inhibitors of autophagosomal formation/function. Knockdown of LC3 expression via siRNA similarly resulted in reduced TzbR cell proliferation and supra-additively interacted with Tzb to re-sensitize TzbR cells. Sub-groups of Tzb-naive SKBR3 parental cells accumulated LC3 punctate structures and decreased p62 expression after treatment with high-dose Tzb, likely promoting their own resistance. This is the first report showing that HER2-overexpressing breast cancer cells chronically exposed to Tzb exhibit a bona fide up-regulation of the autophagic activity that efficiently works to protect breast cancer cells from the growth-inhibitory effects of Tzb. Therapeutic targeting autophagosome formation/function might represent a novel molecular avenue to reduce the emergence of Tzb resistance in HER2-dependent breast carcinomas.     

Histone deacetylase inhibitor LAQ824 down-regulates Her-2 and sensitizes human breast cancer cells to trastuzumab, taxotere, gemcitabine, and epothilone B

Histone deacetylase inhibitors induce hyperacetylation of the amino-terminal lysine residues of the core nucleosomal histones, which results in chromatin remodeling and altered gene expression. Present studies demonstrate that exposure to a novel hydroxamic acid analogue histone deacetylase inhibitor, LAQ824, induced p21WAF1 and p27KIP1 and caused growth arrest and apoptosis of human breast cancer SKBR-3 and BT-474 cells that possess amplification and overexpression of Her-2/neu. Treatment with LAQ824 depleted the mRNA and protein levels of Her-2/neu-encoded Her-2, which was associated with attenuation of pAKT, c-Raf-1, and phosphorylated mitogen-activated protein kinase levels. LAQ824 also induced the acetylation of heat shock protein (hsp) 90, resulting in inhibition of its binding to ATP, which has been shown to impair the chaperone association of hsp 90 with its client proteins, Her-2, AKT, and c-Raf-1. Consistent with this, treatment with LAQ824 shifted the binding of Her-2 from hsp 90 to hsp 70, promoting proteasomal degradation of Her-2. Thus, LAQ824 depletes Her-2 through two mechanisms: attenuation of its mRNA levels and promotion of its degradation by the proteasome. Following LAQ824 treatment, the cell membrane association, autotyrosine phosphorylation, and colocalization of Her-2 with HER-3 also declined. Cotreatment with LAQ824 significantly increased trastuzumab-induced apoptosis of BT-474 and SKBR-3 cells. This was associated with greater attenuation of Her-2, c-Raf-1, and pAKT levels. LAQ824 also enhanced taxotere-induced, epothilone B-induced, and gemcitabine-induced apoptosis of BT-474 and SKBR-3 cells. These findings suggest that LAQ824 is active against human breast cancer cells and has the potential to improve the efficacy of trastuzumab, taxotere, gemcitabine, and epothilone B against breast cancer with Her-2/neuamplification.     

Circ_016719 plays a critical role in neuron cell apoptosis induced by I/R via targeting miR-29c/Map2k6

BackgroundStroke is a leading cause of mortality worldwide. Rac-MAPK kinase 6 (Map2k6) plays important roles in cell proliferation and apoptosis. However, the role played by Map2k6 in stroke injury and the underlying mechanism of action remain unknown.MethodsMice received cerebral ischemia/reperfusion (I/R) injuries by transient middle cerebral artery occlusion. HT22 cells were subjected to oxygen glucose deprivation and reoxygenation (OGD/R) to simulate an I/R injury. Subsequently, the levels of circ_016719, miR-29c and Map2k6 expression were determined, and their interactions were examined by luciferase assays. Circ_016719 knockdown, miR-29c inhibition or Map2k6 overexpression was induced in HT22 cells; after which, the cells were examined for their viability, apoptosis, autophagy and proliferation, as well their levels of Map2k6, p38, p53, LC3B–I, LC3B-II, Beclin 1, and p62 expression.ResultsSignificantly increased levels of circ_016719 and Map2k6, and decreased levels of miR-29c were observed in both in vivo and in vitro I/R injury models. In HT22 cells, circ_016719 knockdown significantly increased miR-29c expression and cell proliferation, but decreased Map2k6 expression and cell apoptosis. Additionally, significant increases in LC3B–I and p62 levels and decreased LC3B-II levels were observed, indicating that circ_016719 knockdown had significantly inhibited autophagy. Furthermore, additional inhibition of miR-29c markedly suppressed the effects of circ_016719 knockdown; however, that suppression was significantly attenuated by Map2k6 overexpression. Additionally, Map2k6 was identified as a direct target of miR-29c, which in turn, might be sponged by circ_016719.ConclusionsOur results suggest that circ_016719 directly targets miR-29c, and thereby regulates the expression and functions of Map2k6, which significantly contributes to the pro-apoptotic role of circ_016719.     

Role of autophagy in cholangiocarcinoma: Pathophysiology and implications for therapy

Cholangiocarcinoma (CCA) is a malignant tumour of the biliary system that originates from the neoplastic transformation of cholangiocytes. CCA is characterized by late diagnosis and poor outcome, with surgery considered as the last option for management. Autophagy is a physiological lysosomal degradation process, essential for cellular homeostasis and ubiquitous in all eukaryotic cells. Several studies have reported a potential involvement of autophagy in cancer, but it remains unclear whether activation of this process represents a survival mechanism of cancer cells. In the present review, we examine the autophagic process and summarize the current knowledge about the involvement of autophagy in the progression of cancer. The link between autophagy and chemoresistance and the use of autophagic markers in diagnosis are also considered in detail. Preliminary evidence shows that the combination of autophagy modulators (activators or inhibitors) with conventional chemotherapeutic agents offers a possible treatment option against signalling pathways that are hyperactivated or altered in CCA. In vitro evidence suggests that combination of chemotherapy agents, such as cisplatin, under activation or inhibition of autophagic processes, in two different CCA cell lines, may improve chemosensitivity and reduce cell survival, respectively. A deeper understanding of these pathways, in both cancer and non-cancer cells, could unveil possible therapeutic targets to treat CCA patients.     

白血病细胞自噬调控的研究进展

自噬是真核细胞内一种保守的自降解系统,参与多种生理病理过程。自噬体是自噬发生的典型特征,针对自噬体形成和降解的调节是自噬的主要调控环节。自噬具有促进细胞存活和死亡的双重特性,在某种程度上,自噬决定了细胞的存亡。自噬与肿瘤的关系十分密切,涉及到肿瘤的发生发展、转移及耐药。靶向自噬有可能成为治疗肿瘤和解决耐药的新策略。随着细胞自噬在白血病研究中的深入,将进一步阐明白血病细胞自噬的诱导和调控机制,为其治疗提供新的靶点和策略,或使白血病治愈成为可能。本文就自噬特征与调控、自噬与肿瘤、自噬与白血病及白血病细胞自噬的调控进行了综述。     

Trans,trans,trans-[PtIV(N3)2(OH)2(py)(NH3)]: A Light-Activated Antitumor Platinum Complex That Kills Human Cancer Cells by an Apoptosis-Independent Mechanism

Photoactivatable Pt(IV) diazido complexes have unusual photobiologic properties. We show here that trans,trans,trans-[Pt(IV)(N(3))(2)(OH)(2)(py)(NH(3))] complex 3 is a potent photoactivated cytotoxin toward human cancer cells in culture, with an average IC(50) value in 13 cell lines of 55 ± 28 μmol/L after 30 minutes (0.12 mW/cm(2)) photoactivation with UVA, although visible light was also effective. Photoactivated complex 3 was noncross-resistant to cisplatin in 3 of 4 resistant cell lines. Cell swelling but very little blebbing was seen for HL60 cells treated with irradiated complex 3. Unlike cisplatin and etoposide, both of which cause apoptosis in HL60 cells, no apoptosis was observed for UVA-activated complex 3 by the Annexin V/propidium iodide flow cytotometry assay. Changes in the levels of the autophagic proteins LC3B-II and p62 in HL60 cells treated with UVA-activated complex 3 indicate autophagy is active during cell death. In a clonogenic assay with the SISO human cervix cancer cell line, 3 inhibited colony formation when activated by UVA irradiation. Antitumor activity of complex 3 in mice bearing xenografted OE19 esophageal carcinoma tumors was photoaugmented by visible light. Insights into the novel reaction pathways of complex 3 have been obtained from (14)N{(1)H} nuclear magnetic resonance studies, which show that photoactivation pathways can involve release of free azide in buffered solution. Density functional theory (DFT) and time-dependent DFT calculations revealed the dissociative character of singlet and triplet excited states of complex 3, which gives rise to reactive, possibly cytotoxic azidyl radicals. Mol Cancer Ther; 11(9); 1894-904. ?2012 AACR.     

Characterization of the cytotoxic activities of novel analogues of the antitumor agent,lavendamycin

Lavendamycin is a bacterially derived quinolinedione that displays significant antimicrobial and antitumor activities. However, preclinical development of lavendamycin as an anticancer agent was halted due to the poor aqueous solubility and relatively nonspecific cytotoxic activity of this compound. In this report, we have examined the cytotoxic activities of a series of novel lavendamycin analogues. The cytotoxic activities of these compounds were evaluated in clonogenic survival assays with A549 lung carcinoma cells. Compounds bearing an amide or amine substituent at the R(3) position were the most potent inhibitors of colony formation. MB-97, the most active member of this subgroup, decreased clonogenic outgrowth by 70% at a concentration of 10 n. Treatment of A549 cells with MB-97 led to an increase in p53 protein expression and phosphorylation and a concomitant increase in the expression of the p53 target gene, p21. Exposure of p53-positive cells to MB-97 triggered cell cycle arrest in G(1) and G(2) phases but induced a selective G(2)-phase arrest in p53-negative cells. MB-97 treatment also induced a higher level of apoptosis in p53-null cells relative to their p53-positive counterparts. Finally, MB-97 showed significant cytotoxic activity in the National Cancer Institute's panel of 60 cancer cell lines and antitumor activity in vivo in hollow fiber tumorigenesis assays.     

Rab27A在4种不同人乳癌细胞株中的表达

目的检测Rab27A在4种人乳癌细胞中的定位、表达情况,初步研究Rab27A表达对乳癌细胞生物学特性的影响。方法采用RT-PCR技术,检测Rab27A mRNA在4种人乳癌细胞MCF-7、MDA-MB-231、MDA-MB-435和MDA-MB-435HM中的表达并进行半定量分析。结果Rab27A mRNA在人乳癌细胞MDA-MB-231、MDA-MB-435及MDA-MB-435HM中的表达水平分别是MCF-7中的2.1、3.4和6.9倍,差异有显著意义（F=17.74～136.23,P〈0.05、0.01）;Rab27A蛋白在人乳癌细胞MDA-MB-231、MDA-MB-435及MDA-MB-435HM中的表达分别是MCF-7中的2.8、4.9和9.2倍,差异有显著性（F=37.74～154.29,P〈0.05、0.01）。Rab27A蛋白弥散性分布于MCF-7、MDA-MB-231和MDA-MB-435细胞浆中,MDA-MB-231和MDA-MB-435中又可见Rab27A蛋白在核周凝聚。结论Rab27A表达可能与乳癌细胞侵袭转移能力有关。     

Bisphosphonates induce autophagy by depleting geranylgeranyl diphosphate

Multiple studies have implicated the depletion of isoprenoid biosynthetic pathway intermediates in the induction of autophagy. However, the exact mechanism by which isoprenoid biosynthesis inhibitors induce autophagy has not been well established. We hypothesized that inhibition of farnesyl diphosphate synthase (FDPS) and geranylgeranyl diphosphate synthase (GGDPS) by bisphosphonates would induce autophagy by depleting cellular geranylgeranyl diphosphate (GGPP) and impairing protein geranylgeranylation. Herein, we show that an inhibitor of FDPS (zoledronate) and an inhibitor of GGDPS (digeranyl bisphosphonate, DGBP) induce autophagy in PC3 prostate cancer and MDA-MB-231 breast cancer cells as measured by accumulation of the autophagic marker LC3-II. Treatment of cells with lysosomal protease inhibitors [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E-64d) and pepstatin A] in combination with zoledronate or digeranyl bisphosphonate further enhances the formation of LC3-II, indicating that these compounds induce autophagic flux. It is noteworthy that the addition of exogenous GGPP prevented the accumulation of LC3-II and impairment of Rab6 (a GGTase II substrate) geranylgeranylation by isoprenoid pathway inhibitors (lovastatin, zoledronate, and DGBP). However, exogenous GGPP did not restore isoprenoid pathway inhibitor-induced impairment of Rap1a (a GGTase I substrate) geranylgeranylation. In addition, specific inhibitors of farnesyl transferase and geranylgeranyl transferase I are unable to induce autophagy in our system. Furthermore, the addition of bafilomycin A1 (an inhibitor of autophagy processing) enhanced the antiproliferative effects of digeranyl bisphosphonate. These results are the first to demonstrate that bisphosphonates induce autophagy. Our study suggests that induction of autophagy in PC3 cells with these agents is probably dependent upon impairment of geranylgeranylation of GGTase II substrates.