Synergistic Antineoplastic Effects Research Articles

MP53-08 METFORMIN USE IS ASSOCIATED WITH IMPROVED SURVIVAL IN VETERANS WITH ADVANCED PROSTATE CANCER ON ANDROGEN DEPRIVATION THERAPY

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) III1 Apr 2017MP53-08 METFORMIN USE IS ASSOCIATED WITH IMPROVED SURVIVAL IN VETERANS WITH ADVANCED PROSTATE CANCER ON ANDROGEN DEPRIVATION THERAPY Kyle Richards, Jinn-ing Liou, Vincent Cryns, Tracy Downs, Jason Abel, and David Jarrard Kyle RichardsKyle Richards More articles by this author , Jinn-ing LiouJinn-ing Liou More articles by this author , Vincent CrynsVincent Cryns More articles by this author , Tracy DownsTracy Downs More articles by this author , Jason AbelJason Abel More articles by this author , and David JarrardDavid Jarrard More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1659AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Metformin is a commonly prescribed glucose-lowering therapy for patients with type 2 diabetes mellitus (T2DM). Basic research supports a synergistic anti-neoplastic effect of metformin for various cancers including prostate cancer (PC). We hypothesize that metformin plus androgen deprivation therapy (ADT) may be synergistic in men with advanced PC. METHODS Using national Veterans Affairs databases, we identified all men diagnosed with PC between 2000-2008 that were treated with ADT and had follow-up through October of 2015. We excluded patients that were treated with ADT for ≤6 months or were receiving ADT concurrently with localized radiation therapy. Three cohorts were identified including non-diabetics, diabetics on metformin, and diabetics not on metformin. Our primary outcome was overall survival (OS) and secondary outcomes included skeletal related events (SRE) and PC-specific survival. RESULTS The total cohort after exclusions consisted of 87,344 patients of which 53,893 (61%) were non-diabetics, 14,517 (17%) were diabetics on metformin and 18,934 (22%) were diabetics not on metformin. Mean age was 75 ±11y (non-diabetics), 71 ±12 (diabetics on metformin), and 75 ±10 (diabetics not on metformin), p<0.001. The median OS was 7.1, 9.1 and 7.4 y respectively (p<0.001). Multivariable Cox proportional hazards analysis assessing for predictors of OS showed improved survival in diabetics on metformin (HR 0.77, 95% CI 0.74-0.81; p<0.001) vs. diabetics not on metformin (HR 0.99, 95% CI 0.95-1.03; p=0.5) with non-diabetics as referent group while controlling for age, co-morbidity, and Gleason score. Assessing for predictors of SRE revealed no association between metformin use (HR 0.99, 95% CI 0.92-1.07; p=0.8) and SRE. Lastly, PC-specific survival was improved in diabetics on metformin (HR 0.72, 95% CI 0.67-0.78; p<0.001) and to a lesser extent diabetics not on metformin (HR 0.87, 95% CI 0.81- 0.93; p<0.001) with non-diabetics as referent group. CONCLUSIONS Metformin use in Veterans with advanced PC receiving ADT is associated with improved OS and cancer-specific survival. Improved outcomes for PC patients receiving metformin should be evaluated in a prospective clinical trial. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e715-e716 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Kyle Richards More articles by this author Jinn-ing Liou More articles by this author Vincent Cryns More articles by this author Tracy Downs More articles by this author Jason Abel More articles by this author David Jarrard More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract A31: eIF4F links translation to energy stress response in cancer

Abstract Protein synthesis is one of the most energy consuming process in the cell. Oncogenic kinases (e.g. EGFR/HER2, BCR/ABL and BRAF) play a central role in reprogramming translation and energy metabolism in neoplasia, whereby cancer cells must provide sufficient ATP to support increased levels of protein synthesis required for neoplastic growth. The downstream mechanisms that link translational machinery and energy homeostasis in cancer, however, remain largely unknown. We found that widely used anti-diabetics (biguanides) abrogate adaptations to EGFR/HER2 inhibitor-induced energetic stress, which results in synergistic anti-neoplastic effects both in vitro and in vivo. In turn, breast cancer cells in which 4E-BP1/2 expression was abrogated by CRISPR were partially resistant to the combination of EGFR/HER2 inhibitors and biguanides. This was paralleled by the inability of the drugs to inhibit the eIF4F complex assembly and translation of mRNAs encoding important metabolic regulators including those involved in serine biogenesis (PHGDH, PSAT1) and one carbon metabolism (MTHFD1L). Comparable results were observed when BRAF and BCR/ABL inhibitors were combined with biguanides, which suggests that translational regulation of metabolic genes via the mTORC1/4E-BE/eIF4E pathway plays a major role in energy stress response in cancer. Together our findings demonstrate that the eIF4F complex is an important mediator of metabolic adaptation in response to the combination of biguanides and clinically-used kinase inhibitors and suggest that the efficiency of such anti-cancer strategies are dependent on the integrity of the translation initiation machinery. Citation Format: Laura Hulea, Marie Cargnello, Simon-Pierre Gravel, Young Im, Shannon McLaughlan, Yunhua Zhao, Jenna Ching, Yutian Cai, Ola Larsson, Michael Ohh, Josie Ursini-Siegel, Julie St-Pierre, Michael Pollak, Ivan Topisirovic. eIF4F links translation to energy stress response in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A31.

Hydroxyurea Induces Growth Inhibition in BCR-ABL1 T315I+ Clones and Synergizes with Ponatinib in Killing TKI-Resistant CML Cells

In chronic myeloid leukemia (CML), the occurrence of BCR-ABL1 T315I is associated with resistance against first- and second-generation BCR-ABL1 tyrosine kinase inhibitors (TKI). Ponatinib is a third generation TKI that exerts strong anti-neoplastic effects in advanced CML and is capable of suppressing the kinase activity of BCR-ABL1 T315I. However, therapy with ponatinib is associated with potentially severe side effects. In addition, resistance against ponatinib may develop in sub-clones carrying multiple (compound) mutations in BCR-ABL1. In addition, BCR-ABL1-independent oncogenic pathways contribute to drug resistance. For these patients, alternative therapies such as stem cell transplantation (SCT) or various drug combinations are often considered.Hydroxyurea (HU) is used for initial or palliative cytoreduction in CML. However, the effects of HU on TKI-resistant mutant sub-clones have not been examined so far. The aims of this study were to explore the effects of HU on CML clones carrying BCR-ABL1 T315I as individual mutation or in compound-context, and to investigate anti-leukemic effects of the drug combination ponatinib+HU. In in vitro studies, primary patient-derived cells, human CML cell lines (K562, KU812, KCL-22), and Ba/F3 cells expressing wild type (wt) BCR-ABL1, BCR-ABL1 T315I, or BCR-ABL1 compound mutants involving T315 were examined. Cell proliferation was quantified by measuring 3H-thymidine uptake. Drug effects on competitive clonal growth were analyzed by mixing two Ba/F3 clones, one expressing BCR-ABL1 T315I with GFP and one BCR-ABL1 T315I/E255V labeled by tdTomato, at a 1:1 ratio. Then, cells were exposed to HU, pontinib, or HU+ponatinib for 72 hours, and the percentage of viable cells in each clone was analyzed by flow cytometry. The in vivo response of primary CML cells carrying BCR-ABL1 T315I to HU was examined in 4 TKI-resistant CML patients who were treated with HU (1-3 g/day) for up to 18 months. In these patients, we measured white blood counts (WBC), differential counts, and BCR-ABL1 transcript levels in peripheral blood (PB) by qPCR. The percentage of BCR-ABL1 T315I compared to total BCR-ABL1 was determined by ligation-dependent PCR. In all 4 patients treated with HU, WBC and total BCR-ABL1 mRNA levels remained stable for 3-12 months. Surprisingly, in 3 of 4 patients, the leukemic sub-clone expressing BCR-ABL1 T315Iwas no longer detectable after HU-treatment. After 3 months, 2/4 patients received allogenic SCT. In the other 2 patients, the disease remained stable for 6 and 12 months, respectively. In our in vitro studies, HU was found to inhibit the growth of all BCR-ABL1+ cell lines, including K562 (IC50: 1120±89 µM), KU812 (IC50: 216±32 µM), and KCL-22 (IC50: 196±23 µM) as well as Ba/F3 cells harboring BCR-ABL1 T315I as single mutation (IC50: 74±25 µM) or as compound together with E255V (IC50: 86±2 µM), F311L (IC50: 76±20 µM), F359V (IC50: 69±10 µM), or G250E (IC50: 89±4 µM). Interestingly, Ba/F3 cells exhibiting BCR-ABL1 T315I alone or in compound configuration were more sensitive to HU compared to Ba/F3 cells expressing wt BCR-ABL1 (IC50: 236±49 µM). As expected, HU was also found to inhibit growth of primary CML cells. In subsequent experiments, HU and ponatinib were found to synergize with each other in inhibiting growth of K562, KU812, and KCL-22 cells as well as Ba/F3 cells carrying BCR-ABL1 T315I (Figure) or BCR-ABL1-T315I/F359V. In cell mix experiments, ponatinib exerted strong growth-inhibitory effects on Ba/F3-T315I cells but not on Ba/F3-T315I/E255V cells, whereas HU was found to produce stronger effects on Ba/F3-T315I/E255V cells, and only the combination of both drugs resulted in complete suppression of both cell lines. In conclusion, HU exerts strong sub-clone-specific anti-neoplastic effects in TKI-resistant CML cells, both in patients with BCR-ABL1 T315I+CML and in various cell line models, including sub-clones harboring BCR-ABL1 T315I as single mutation or in compound configuration. In addition, we show that HU and ponatinib produce strong synergistic anti-neoplastic effects on TKI resistant CML cells, including sub-clones carrying T315I. These observations may have clinical implications and may pave the way for more effective sub-clone-eradicating but also palliative or bridging-to-SCT concepts in advanced CML. Clinical studies are now warranted to define the exact value of the drug combination ponatinib+HU in TKI-resistant CML. [Display omitted] DisclosuresSperr:Novartis: Honoraria; Amgen: Honoraria, Research Funding. Lion:Ariad: Honoraria; Amgen: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Hoermann:Ariad: Honoraria; Novartis: Honoraria; Gilead: Research Funding; Amgen: Honoraria. Deininger:CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Valent:Amgen: Honoraria; Novartis: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding; Ariad: Honoraria, Research Funding.

Anticoagulation in combination with antiangiogenesis and chemotherapy for cancer patients: evidence and hypothesis.

Hypercoagulable state and disorganized angiogenesis are two conspicuous characteristics during tumor progression. There are a considerable number of clinical trials focusing on the effects of anticoagulant and antiangiogenic drugs on the survival of cancer patients. Favorable outcomes have been observed. Excessive blood coagulation not only causes cancer-associated thrombosis, which is a common complication and is the second leading cause of death in patients, but also decreases intratumoral perfusion rates and drug delivery by reducing the effective cross-sectional area of blood vessels. Meanwhile, structural and functional abnormalities of the tumor microvasculature also compromise convective drug transport and create a hypoxic and acidic microenvironment. Vascular normalization strategy can temporarily recover the abnormal state of tumor vasculature by improving blood density, dilation, and leakiness, resulting in enhanced penetration of chemotherapies and oxygen within a short time window. In this article, we first review the evidence to support the opinion that anticoagulant and antiangiogenic therapy can improve cancer survival through several underlying mechanisms. Next, we speculate on the feasibility and value of the combined strategy and discuss whether such a combination has a synergistic antineoplastic effect in cancer patients by way of increasing blood vessel perfusion and drug distribution.

Identification of CD25 as STAT5-Dependent Growth Regulator of Leukemic Stem Cells in Ph+ CML.

In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs. CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25(+)CML cell line KU812. In contrast to normal hematopoietic stem cells, CD34(+)/CD38(-)CML LSCs expressed the IL-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin(-)/Sca-1(+)/Kit(+)stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5 depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cellsin vitroand their engraftmentin vivoin NOD/SCID-IL-2Rγ(-/-)mice. In drug-screening experiments, the PI3K/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic antineoplastic effects on CML cells when applied in combination with nilotinib or ponatinib. CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth regulator of CML LSCs, which may have biologic and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML.

Epigenetic therapy of acute myeloid leukemia using 5-aza-2'-deoxycytidine (decitabine) in combination with inhibitors of histone methylation and deacetylation.

BackgroundThe silencing of tumor suppressor genes (TSGs) by aberrant DNA methylation occurs frequently in acute myeloid leukemia (AML). This epigenetic alteration can be reversed by 5-aza-2’-deoxcytidine (decitabine, 5-AZA-CdR). Although 5-AZA-CdR can induce complete remissions in patients with AML, most patients relapse. The effectiveness of this therapy may be limited by the inability of 5-AZA-CdR to reactivate all TSGs due to their silencing by other epigenetic mechanisms such as histone methylation or chromatin compaction. EZH2, a subunit of the polycomb repressive complex 2, catalyzes the methylation of histone H3 lysine 27 (H3K27) to H3K27me3. 3-Deazaneplanocin-A (DZNep), an inhibitor of methionine metabolism, can reactivate genes silenced by H3K27me3 by its inhibition of EZH2. In a previous report, we observed that 5-AZA-CdR, in combination with DZNep, shows synergistic antineoplastic action against AML cells. Gene silencing due to chromatin compaction is attributable to the action of histone deacetylases (HDAC). This mechanism of epigenetic gene silencing can be reversed by HDAC inhibitors such as trichostatin-A (TSA). Silent TSGs that cannot be reactivated by 5-AZA-CdR or DZNep have the potential to be reactivated by TSA. This provides a rationale for the use of HDAC inhibitors in combination with 5-AZA-CdR and DZNep to treat AML.ResultsThe triple combination of 5-AZA-CdR, DZNep, and TSA induced a remarkable synergistic antineoplastic effect against human AML cells as demonstrated by an in vitro colony assay. This triple combination also showed a potent synergistic activation of several key TSGs as determined by real-time PCR. The triple combination was more effective than the combination of two agents or a single agent. Microarray analysis showed that the triple combination generated remarkable changes in global gene expression.ConclusionsOur data suggest that it may be possible to design a very effective therapy for AML using agents that target the reversal of the following three epigenetic “lock” mechanisms that silence gene expression: DNA methylation, histone methylation, and histone deacetylation. This approach merits serious consideration for clinical investigation in patients with advanced AML.

BRAF inhibition improves tumor recognition by the immune system

In spite of the fact that they occur at high rates, the clinical responses of BRAFV600 mutant metastatic melanoma to BRAF inhibitors are usually short-lasting, with most cases progressing within less than 8 mo. Immunomodulatory strategies initiated after progression have recently been reported to be poorly efficient. By characterizing the immunological interactions between T cells and cancer cells in clinical material as well as the influence of the FDA-approved BRAF inhibitor vemurafenib on the immune system, we aimed at unraveling new strategies to expand the efficacy of adoptive T-cell transfer, which represents one of the most promising approaches currently in clinical development for the treatment of metastatic melanoma. Here we show that blocking the BRAF-MAPK pathway in BRAF signaling-addicted melanoma cells significantly increases the ability of T cells contained in clinical grade tumor-infiltrating lymphocytes to recognize autologous BRAFV600 mutant melanoma cell lines in vitro. Antitumor reactivity was improved regardless of the class of antigen recognized by tumor-specific CD8+ T cells. Microarray data suggests that improved tumor recognition is associated with modified expression of MHC Class I-associated proteins as well as of heat-shock proteins. In conclusion, our preclinical data suggest that an appropriately timed sequential treatment of BRAFV600 mutant melanoma with vemurafenib and adoptive T-cell transfer might result in synergistic antineoplastic effects owing to an increased immunogenicity of cancer cells.

Azacytidine and erlotinib exert synergistic effects against acute myeloid leukemia

The term myelodysplastic syndrome (MDS) identifies a heterogeneous group of clonal disorders originating from bone marrow stem cells that often progress to acute myeloid leukemia (AML). The reference treatments for MDS include the DNA methyltransferase inhibitors azacytidine and decitabine. Recently, the epidermal growth factor receptor (EGFR) inhibitor erlotinib has been shown to exert antileukemic activity in vitro and in vivo, independent of the EGFR. Thanks to this feature, erlotinib is currently being tested as an antileukemic drug in clinical trials. Here, we report that azacytidine and erlotinib mediate synergistic antineoplastic effects in several primary or secondary (post-MDS) AML cell lines. The combination of azacytidine and erlotinib blocked cell-cycle progression and induced caspase-dependent apoptosis more consistently than either of the two agents alone. These effects were not a consequence of cellular differentiation and could be discriminated from each other, as the former depended on caspases whereas the latter did not. The synergy between azacitidine and erlotinib, which involved the proteasomal degradation of the anti-apoptotic Bcl-2 family members MCL-1 and BCL2L10 and the upregulation of their pro-apoptotic counterpart PUMA, was abolished when azacytidine was replaced by decitabine but persisted when erlotinib was substituted with gefitinib, another EGFR inhibitor. Of note, the intracellular accumulation of azacytidine was exacerbated by both erlotinib and gefitinib, pointing to a pharmacokinetic mechanism of synergy. In approximately half of the cases studied, marrow and circulating blasts from MDS and AML patients, respectively, exhibited hyperadditive cytotoxic responses to the combination of azacytidine and erlotinib. These results strongly suggest that the combination of azacytidine and erlotinib may exert clinically relevant antileukemic effects.

Probenecid is a chemosensitizer in cancer cell lines

Resistance and toxicity are the major barriers to successful cancer chemotherapies. Developing molecules that reduce drug resistance and improve antineoplastic effects is of great interest for cancer research; ideally, these substances should not affect the pharmacodynamics of the chemotherapeutic agent while providing a synergistic antineoplastic effect. In this study, we tested in vitro co-administration of the antineoplastic agents cisplatin or paclitaxel with probenecid, an anion channel inhibitor, in a panel of cancer cell lines to determine the cytotoxicity and synergistic effects of these drug combinations. In addition, we measured the clonogenicity and apoptotic index in these cells. We observed a synergistic interaction between probenecid and the chemotherapeutic agents, and increasing doses of probenecid resulted in a significant decrease in the effective doses of the chemotherapeutic agents. For the antineoplastic agent and probenecid combinations, we found increased cell death, reduced colony formation, and a higher number of apoptotic cells, compared with treatment of cisplatin or paclitaxel alone. Further research is necessary to elucidate the molecular mechanisms by which the synergistic effect occurs. If these synergistic effects can be reproduced in vivo, the co-administration of probenecid with different chemotherapeutic agents may provide a valid treatment in patients with chemotherapy resistance.

Zytoreduktive Chirurgie und hypertherme intrathorakale Chemotherapieperfusion

Recently cytoreductive surgery of primary and secondary pleural tumors has been combined with hyperthermic intrathoracic chemotherapy perfusion (HITHOC) for better local tumor control. In comparison to simple instillation of chemotherapeutic agents into the pleural cavity, the combination of surgical resection of pleural tumors and simultaneous HITHOC seems to be a more effective treatment. Intra-operative perfusion allows an improved distribution of the drug in the pleural space and a higher local concentration of the chemotherapeutic agents in contrast to systemic chemotherapy. Additional advantages of HITHOC are a better response to chemotherapeutic agents and synergistic antineoplastic effects. A prerequisite for safe application of HITHOC is compliance with safety regulations. Due to the reduction in morbidity and mortality this new concept is a valuable alternative for selected patients who do not undergo radical resection (e.g. extrapleural pneumonectomy). HITHOC is an additional therapeutic option in the multimodal treatment of patients with primary or secondary tumors of the pleura.

Synergistic anti-neoplastic effect of AG1478 in combination with cisplatin or paclitaxel on human endometrial and ovarian cancer cells

AG1478, a potent inhibitor of epidermal growth factor receptor (EGFR), facilitates the induction of cell death in combination with a variety of cytotoxic and chemotherapeutic agents in certain human tumor cell lines. The purpose of this study was to elucidate the effect of AG1478 on three endometrial cancer and two ovarian cancer cell lines as compared to normal human endometrial epithelial cells. Cells were treated with various concentrations of AG1478 alone or in combination with the chemotherapeutic drugs cisplatin or paclitaxel, and the effect of AG1478 on cell growth, the cell cycle and apoptosis was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that all the cancer cell lines were sensitive to the growth-inhibitory effect of AG1478. Normal endometrial epithelial cells remained viable after treatment with AG1478 at the same doses as those which induced growth inhibition in the endometrial and ovarian cancer cells. Synergistic anti-neoplastic effects were obtained with a combination of AG1478 and cytostatic drugs. Cell-cycle analysis indicated that exposure to these drugs decreased the proportion of cells in the S-phase and increased the proportion in the sub G0/G1 fractions of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and by loss of mitochondrial transmembrane potential. These results suggest that AG1478 alone or in combination with chemotherapeutic drugs may be a novel therapeutic option for the treatment of endometrial and ovarian cancer.

Synergistic antineoplastic effect of DLC1 tumor suppressor protein and histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), on prostate and liver cancer cells: Perspectives for therapeutics

Inactivation of tumor suppressor genes is a major contributing alteration in the initiation or progression of cancer. The human tumor suppressor gene DLC1 (deleted in liver cancer 1) is frequently downregulated or silenced in multiple cancers, predominantly by epigenetic mechanisms. With the current considerable interest and progress in epigenetic therapy, a number of promising antineoplastic agents, particularly histone deacetylase (HDAC) inhibitors, have been developed and used successfully in clinical trials. Both DLC1 and HDAC inhibitors exert antineoplastic functions, and their combined action could be exploited for a more effective cancer therapy. To evaluate the potential benefits of this approach, we examined the antineoplastic effects of adenoviral (Ad)-DLC1-mediated transduction and exposure to suberoylanilide hydroxamic acid (SAHA), a powerful HDAC inhibitor, in two human cancer cell lines that lack intrinsic DLC1 expression, 22Rv1 prostate cancer cells and 7703K human hepatocellular carcinoma cells. Consistent with the oncosuppressive function of DLC1 in several cancers, including prostate and liver cancer, transduction of 22Rv1 and 7703K cells with an Ad-DLC1 expression vector resulted in alterations of cell morphology, induction of apoptosis, and inhibition of cell proliferation, migration, and anchorage-independent growth. A low concentration of SAHA (5 microM) efficiently restored the expression of DLC1 in 22Rv1 cells that lack DLC1 expression due to histone deacetylation but had a minimal effect in 7703K cells in which silencing of the DLC1 gene is due mainly to promoter hypermethylation. Regardless of the epigenetic mechanism of DLC1 inactivation, SAHA treatment of DLC1-transduced cells had a synergistic inhibitory effect on tumor cell proliferation and tumorigenesis in both cell lines. In 22Rv1 cells, this combination regimen nearly abolished the formation of colonies in semisolid media as a measure of tumorigenicity in vitro. Current in vitro results validate this protocol as a potentially new therapeutic option in certain cancers.

PK11195 enhances chemosensitivity to cisplatin and paclitaxel in human endometrial and ovarian cancer cells

The potential anticancer agent 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a translocator protein ligand, facilitates the induction of cell death by a variety of cytotoxic and chemotherapeutic agents in various human tumor cell lines. The purpose of this study was to elucidate the effect of PK11195 on three endometrial cancer cell lines, two ovarian cancer cell lines and normal human endometrial epithelial cells. Endometrial and ovarian cancer cells were treated with various concentrations of PK11195 alone or in combination with chemotherapeutic drugs (cisplatin, paclitaxel), and its effect on cell growth, the cell cycle, apoptosis and related measurements was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that all endometrial and ovarian cancer cell lines were sensitive to the growth-inhibitory effect of PK11195, although normal endometrial epithelial cells were viable after treatment with the same doses of PK11195 that induced growth inhibition in endometrial and ovarian cancer cells. Synergistic anti-neoplastic effects were obtained by a combination of PK11195 with cytostatic drugs. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to apoptosis. These results suggest that PK11195 alone or in combination with chemotherapeutic drugs might be a new therapeutic option for the treatment of endometrial and ovarian cancers.

Peripheral benzodiazepine receptor ligands induce apoptosis and cell cycle arrest in human hepatocellular carcinoma cells and enhance chemosensitivity to paclitaxel, docetaxel, doxorubicin and the Bcl-2 inhibitor HA14-1

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer deaths worldwide. Thus, novel therapies are urgently needed. A promising approach is the use of peripheral benzodiazepine receptor (PBR) ligands which inhibit the proliferation of various tumors. PBR expression both in human HCC cell lines and in tumor specimens of HCC patients was analyzed by RT-PCR and immunostaining. To evaluate PBR ligands for the treatment of HCC, we tested their effects on human HCC cells. PBR was localized to the mitochondria both of HCC cell lines and tumor tissues of HCC patients. In contrast, normal liver did not express PBR. PBR ligands inhibited the proliferation of HCC cell lines by inducing apoptosis and cell cycle arrest. Apoptosis was characterized by a breakdown of the mitochondrial membrane potential, caspase-3 activation and nuclear degradation. Furthermore, pro-apoptotic Bax was overexpressed while anti-apoptotic Bcl-2 and Bcl-X(L) were suppressed. Cell cycle was arrested both at the G1/S- and G2/M-checkpoints. Synergistic anti-neoplastic effects were obtained by a combination of PBR ligands with cytostatic drugs (paclitaxel, docetaxel, doxorubicin), or with an experimental Bcl-2 inhibitor. This is the first report on the induction of apoptosis and cell cycle arrest by PBR ligands in HCC cells. Moreover, PBR ligands sensitized HCC cells to taxans and doxorubicin.

Synergistic antineoplastic action of DNA methylation inhibitor 5-AZA-2'-deoxycytidine and histone deacetylase inhibitor depsipeptide on human breast carcinoma cells.

During tumorigenesis, cancer-related genes can be silenced by aberrant DNA methylation and by changes in chromatin structure. It has been reported that 5-aza-2'-deoxycytidine, a potent inhibitor of DNA methylation, in combination with histone deacetylase inhibitors, can produce a synergistic reactivation of these genes. The aim of our study was to investigate the in vitro antineoplastic activity of 5-aza-2'-deoxycytidine in combination with depsipeptide, a potent histone deacetylase inhibitor, against MDA-MB-231 and MDA-MB-435 human breast carcinoma cell lines. We observed that the combination of 5-aza-2'-deoxycytidine and depsipeptide produced a synergistic antineoplastic effect against these tumor cells as compared to either agent administered alone. We also investigated the effect of this drug combination on the activation of maspin and gelsolin expression. These 2 genes whose function is to suppress tumor metastasis have been reported to be silenced by epigenetic events in breast cancer. Using semi-quantitative RT-PCR, we observed that 5-aza-2'-deoxycytidine in combination with depsipeptide produced a greater reactivation of both maspin and gelsolin as compared to each agent alone. The synergistic interaction between 5-aza-2'-deoxycytidine and depsipeptide on breast carcinoma cell lines provides a rationale to investigate this interesting drug combination in future clinical trials on patients with advanced breast cancer.

Cytochrome c release is upstream to activation of caspase-9, caspase-8, and caspase-3 in the enhanced apoptosis of anaplastic thyroid cancer cells induced by manumycin and paclitaxel.

We previously demonstrated that the combination of a farnesyltransferase inhibitor, manumycin A, and paclitaxel had a synergistic antineoplastic effect on anaplastic thyroid cancer. In this study we investigated the apoptosis pathway involved. In ARO and KAT-4 cells, manumycin- plus paclitaxel-induced DNA fragmentation was blocked by the inhibitors of caspase-9, caspase-8, and caspase-3. The drug combination enhanced the activation of caspase-9, caspase-8, and caspase-3 and cytochrome c release into the cytosol. Cytochrome c release was not affected by the inhibitors of caspase-9, caspase-8 and caspase-3. In a cell-free reconstitution assay, DNA fragmentation occurred after incubating nuclei purified from untreated KAT-4 cells with deoxy-ATP, exogenous cytochrome c and S-100 extracts from control KAT-4 cells, and also after incubation of purified KAT-4 nuclei with S-100 extracts from KAT-4 cells treated with manumycin-plus-paclitaxel. In both cases, the DNA fragmentation was blocked by the inhibitors of caspase-9, caspase-8 and caspase-3. We concluded that the cytochrome c release was upstream of the activation of caspase-9, caspase-8, and caspase-3 in the enhanced apoptosis of anaplastic thyroid cancer cells treated with manumycin plus paclitaxel, and that the interaction between manumycin and paclitaxel occurred at or upstream of cytochrome c in the apoptosis regulatory pathway in anaplastic thyroid cancer cells.

Exisulind, a selective apoptotic antineoplastic drug

Exisulind (Aptosyn™, Cell Pathways, Inc.) is the first of a new class of targeted, pro-apoptotic drugs that show promise in the treatment of cancer. These agents induce apoptosis (i.e., programmed cell death) in a broad range of pre-cancerous and cancerous tissues without affecting normal cells. The antineoplastic effect of exisulind appears to be the result of activation of protein kinase G (PKG) which leads to multiple downstream effects culminating in apoptosis. Exisulind has demonstrated antineoplastic activity in solid tumour and haematological cancer cell lines and is an inhibitor of tumour growth in rodent models of colon, prostate, bladder, mammary and lung cancer. Preclinical data evaluating selective apoptotic antineoplastic drugs (SAANDs) in combination with various chemotherapy drugs indicates additive or synergistic antineoplastic effects. In clinical studies, exisulind prevented colorectal polyp formation in patients with familial adenomatous polyposis (FAP) over 24 months. In a randomised, placebo-controlled study of prostate cancer patients, exisulind inhibited the rise of prostate-specific antigen (PSA) in men with PSA progression after radical prostatectomy. Exisulind has been well-tolerated by most patients in clinical trials. In conclusion, preclinical evidence and early clinical results suggest that exisulind and other drugs in this class may have wide applications in treating cancer both as monotherapy and in combination with chemotherapy and other targeted agents.

Activation of the retinoic acid receptor beta gene by 5-aza-2'-deoxycytidine in human DLD-1 colon carcinoma cells.

We previously reported that 5-aza-2'-deoxycytidine (5-Aza-CdR) in combination with all-trans retinoic acid (ATRA) produced a synergistic antineoplastic effect on DLD-1 colon carcinoma cells. We also observed that 5-Aza-CdR, a potent inhibitor of DNA methylation, increased the expression of retinoic acid receptor (RAR)-beta. Methylation of cytosine in the promoter-first exon region of genes has been reported to silence their expression. In an attempt to clarify the mechanism responsible for the activation of the RAR-beta gene by 5-Aza-CdR in DLD-1 colon carcinoma cells, we investigated its methylation state by Southern blotting. Our results indicate that DNA hypermethylation of the RAR-beta gene, a putative tumor suppressor gene, may be the mechanism of silencing its expression in these tumor cells. We also reported that a different schedule of 5-Aza-CdR and ATRA produced a synergistic antineoplastic effect on the colon carcinoma cells.

Zidovudine may have synergistic antineoplastic effects

Zidovudine may have synergistic antineoplastic effects

A Phase I–II Study on the Toxicity and Therapeutic Efficacy of 5-Fluorouracil in Combination with Leucovorin and Cisplatinum in Patients with Advanced Colorectal Carcinoma

5-Fluorouracil (5-FU) has been the treatment of choice for colorectal carcinoma with an overall response rate of about 20%. Recent studies have shown that folate (LV) can increase 5-FU therapeutic efficacy, achieving about a 40% response rate without a clear impact on survival. Cisplatinum (CDDP) is usually inactive in colorectal carcinoma, but the association with 5-FU results in a synergistic antineoplastic effect. A phase I-II study was done to assess the maximally tolerated dose (MTD) of CDDP in association with 5-FU + LV. The MTD for CDDP was 20 mg/m2/wk in association with 5-FU 400-500 mg/m2/wk and LV 500 mg/m2/wk. WHO criteria were used for evaluation of both toxicity and response. In the phase I part we found that the main side-effect in 27 evaluable patients (pts) was gastrointestinal toxicity, mainly in the form of nausea/vomiting (92%) and diarrhea (70%) which caused one therapy-related death. Renal (26%) and marrow (59%) toxicity were acceptable. In the phase II part of the study 1 out of 19 evaluable pts (5%) had a complete response of 309 days, 3 pts achieved a partial response (16%) with a median duration od 410 days, 2 pts had a minimal response (10%) with a median duration of 261 days, and 8 pts experienced no change (42%) with a mean duration of 196 + days. In our opinion the 21% response rate obtained in this series is not satisfactory. Nevertheless the very high number of minimal response + no change patients together with the interesting impact on survival in responders may suggest further phase II-III studies.