Gulshan Ara

Activity of PXD101, a histone deacetylase inhibitor, in preclinical ovarian cancer studies

Histone deacetylase inhibitors represent a promising new class of anticancer agents. In the current investigation, we examined the activity of PXD101, a potent histone deacetylase inhibitor, used alone or in combination with clinically relevant chemotherapeutics (docetaxel, paclitaxel, and carboplatin), in preclinical in vitro and in vivo models of ovarian cancer. In vitro activity was examined in ovarian cancer and multidrug-resistant cell lines grown in monolayer culture, and in primary clinical ovarian cancer specimens grown in three-dimensional organoid culture. PXD101 was found to inhibit in vitro cancer cell growth at sub- to low micromolar IC50 potency, exhibited synergistic activity when used in combination with relevant chemotherapeutics, and effectively inhibited the growth of multidrug-resistant cells. In vivo, PXD101 displayed single-agent antitumor activity on human A2780 ovarian cancer s.c. xenografts which was enhanced via combination therapy with carboplatin. In support of these findings, PXD101 was shown to increase the acetylation of α-tubulin induced by docetaxel and the phosphorylation of H2AX induced by carboplatin. Taken together, these results support the clinical evaluation of PXD101 used alone or in combination therapy for the treatment of ovarian cancer. [Mol Cancer Ther 2006;5(8):2086–95]

STRATEGIES FOR SELECTIVE CANCER PHOTOCHEMOTHERAPY: ANTIBODY‐TARGETED and SELECTIVE CARCINOMA CELL PHOTOLYSIS*

Abstract A principle objective in chemotherapy is the development of modalities capable of selectively destroying malignant cells while sparing normal tissues. One new approach to selective photochemo‐therapy, antibody‐targeted photolysis (ATPL) uses photosensitizers (PS) coupled to monoclonal antibodies (MAbs) which bind to eel) surface antigens on malignant cells. Selective destruction of human T leukemia cells (HBP‐ALL) was accomplished by coupling the efficient PS chlorin e6 to an anti‐T cell MAb using dextran carriers. Conjugates with chlorin : MAb ratios of 30 : 1 retained > 85% MAb binding activity, and had a quantum yield for singlet oxygen production of 0.7 ±0.1, the same as that of free chlorin e6. Cell killing was dependent on the doses of both MAb‐PS and 630‐670 nm light, and occurred only in target cell populations which bound the MAb. On the order of 1010 singlet oxygen molecules were necessary to kill a cell.

Comparison of several antiangiogenic regimens alone and with cytotoxic therapies in the Lewis lung carcinoma

Abstract The efficacy of several potential antiangiogenic agents, TNP-470, minocycline, suramin, genistein, interferon δ4, 14(sulfated)-β-cyclodextrin and tetrahydrocortisol, alone and in combination with cytotoxic therapies was examined against primary and metastatic Lewis lung carcinoma. The antiangiogenic agents when administered as single agents or in two-agent combinations were only modestly active as antitumor agents. Three antiangiogenic agent combinations, TNP-470/minocycline, TNP-470/14(SO4)βCD/THC and minocycline/14(SO4)βCD/THC, produced significant increases in tumor growth delay and decreases in the number of lung metastases when administered along with cyclophosphamide compared with cyclophosphamide alone. Two antiangiogenic agent combinations, minocycline/interferon δ4 and minocycline/14 (SO4)βCD/THC, produced significant decreases in the number of lung metastases when administered alone with adriamycin compared with adriamycin alone. The antiangiogenic combinations of TNP-470/minocycline, TNP-470/suramin, TNP-470/genistein, TNP-470/interferon δ4 and TNP-470/14(SO4)βCD/THC, resulted in increased tumor growth delays when administered along with CDDP, BCNU, fractionated radiation or 5-fluorouracil. There was not always a direct correlation between the antiangiogenic regimen that was most beneficial against the primary tumor as compared with disease metastatic to the lungs. These studies establish that a broad range of antiangiogenic therapies can interact in a positive manner with cytotoxic therapies.

Cyclooxygenase and lipoxygenase inhibitors as modulators of cancer therapies

Like many clinical non-small-cell lung cancers, the Lewis lung carcinoma produces prostaglandins. The Lewis lung carcinoma was used as a model of both primary and metastatic disease to assess the ability of cyclooxygenase inhibitors (mefenamic acid, diflunisal, sulindac, and indomethacin), the collagenase inhibitor minocycline, and the lipoxygenase inhibitor phenidone to act as modulators of cytotoxic cancer therapies. Although none of the single modulators given i.p. daily on days 4–18 altered tumor growth or the number of metastases found on day 20, modulator combinations consisting of minocycline/a cyclooxygenase inhibitor and, especially, of phenidone/a cyclooxygenase inhibitor resulted in modest tumor growth delay and a decreased number of lung metastases on day 20. The most effective modulators of cisplatin (CDDP) were phenidone/sulindac and phenidone/indomethacin, which led to 2.4- to 2.5-fold increases in the tumor growth delay produced by CDDP. The most effective modulations of cyclophosphamide resulted from administration of minocycline, minocycline/sulindac, or phenidone/sulindac and led to 2.0- to 2.1-fold increases in tumor growth delay by cyclophosphamide. The most effective modulators of melphalan produced 4.5- to 4.7-fold increases in tumor growth delay by the drug and were minocycline/sulindac, minocycline/mefenamic acid, and phenidone/sulindac. The most effective modulation of carmustine (BCNU) was obtained with minocycline/sulindac and minocycline/diflunisal leading to 2.8- to 3.1-fold increases in tumor growth delay by BCNU. Finally, the most effective modulation of radiation was obtained with minocycline/sulindac and phenidone/sulindac and resulted in 2.8- to 3.3-fold increases in tumor growth delay by radiation. The modulator combination that along with the cytotoxic therapies was most effective against metastatic disease was phenidone/mefenamic acid. There was no clear relationship between effective modulation of the cancer therapies and the degree of reduction in serum levels of prostaglandin E2 and leukotriene B4 by the agents in Lewis lung tumor bearing mice.

Nuclear factor-κB p65 small interfering RNA or proteasome inhibitor bortezomib sensitizes head and neck squamous cell carcinomas to classic histone deacetylase inhibitors and novel histone deacetylase inhibitor PXD101

Histone deacetylase inhibitors (HDI) can inhibit proliferation and enhance apoptosis in a wide range of malignancies. However, HDIs show relatively modest activity in head and neck squamous cell carcinomas (HNSCC), in which we have shown the activation of nuclear factor-κB (NF-κB; NF-κB1/RelA or p50/p65), a transcription factor that promotes expression of proliferative and antiapoptotic genes. In this study, we examined if HDIs enhance activation of NF-κB and target genes and if genetic or pharmacologic inhibition of NF-κB can sensitize HNSCC to HDIs. Limited activity of classic HDIs trichostatin A and sodium butyrate was associated with enhanced activation of NF-κB reporter activity in a panel of six HNSCC cell lines. HDIs enhanced NF-κB p50/p65 DNA binding and acetylation of the RelA p65 subunit. Transfection of small interfering RNAs targeting p65 strongly inhibited NF-κB expression and activation, induced cell cycle arrest and cell death, and further sensitized HNSCC cells when combined with HDIs. The p65 small interfering RNA inhibited HDI-enhanced expression of several NF-κB–inducible genes implicated in oncogenesis of HNSCC, such as p21, cyclin D1, and BCL-XL. Bortezomib, an inhibitor of proteasome-dependent NF-κB activation, also increased sensitization to trichostatin A, sodium butyrate, and a novel HDI, PXD101, in vitro, and to the antitumor effects of PXD101 in bortezomib-resistant UMSCC-11A xenografts. However, gastrointestinal toxicity, weight loss, and mortality of the combination were dose limiting and required parenteral fluid administration. We conclude that HDI-enhanced NF-κB activation is one of the major mechanisms of resistance of HNSCC to HDIs. The combination of HDI and proteasome inhibitor produced increased antitumor activity. Low starting dosages for clinical studies combining HDIs with proteasome inhibitors and IV fluid support may be warranted. [Mol Cancer Ther 2007;6(1):37–50]

Activity of the histone deacetylase inhibitor belinostat (PXD101) in preclinical models of prostate cancer

Histone deacetylase inhibitors (HDACi) represent a promising new class of anticancer agents. In the current investigation, we examined the activity of the HDACi belinostat in preclinical models of prostate cancer. In vitro proliferation assays demonstrated that belinostat potently inhibited the growth of prostate cancer cell lines (IC50 < 1.0 μM) and was cytotoxic to these cells. Washout experiments indicated that exposure to belinostat for relatively short periods of time (<12 hr) induced suboptimal growth‐inhibition and that cells exposed to 1.0 μM belinostat for 48 hr retained the capacity for regrowth following drug withdrawal, while cells exposed to 4.0 μM belinostat were irreversibly growth‐inhibited. Cell cycle analyses demonstrated that belinostat induced G2/M arrest and increased the percentage of cells with subG1 DNA content, thus confirming the growth‐inhibitory and cytotoxic effects of this compound. Normal prostate epithelial cells were generally less susceptible to the effects of belinostat than were prostate cancer cells. In an orthotopic prostate cancer tumor model, belinostat inhibited tumor growth by up to 43%. Moreover, metastatic lung lesions were present in 47% of vehicle‐treated animals but in none of the animals administered belinostat. Consistent with its observed antimetastatic activity, belinostat inhibited the migration of prostate tumor cells and increased the production of tissue inhibitor of metalloproteinase‐1 (TIMP‐1) by these cells, the latter effect being replicated by siRNA knockdown of HDAC3. Belinostat also increased the expression of p21 and decreased the expression of potentially oncogenic proteins (mutant p53 and ERG). These results support the clinical evaluation of belinostat for the treatment of prostate cancer.

Sequence effect of irinotecan (CPT-11) and topoisomerase II inhibitors in vivo

Abstract The DNA topoisomerases I and II are the target of several clinically important antineoplastic agents which produce DNA cleavage by stabilization of the covalent DNA-protein bond with resultant cell death after DNA synthesis is attempted. Depletion of the target topoisomerase and reciprocal changes in the other occur with drug treatment. Purpose and methods: To develop empiric treatment regimens of combinations and sequences of agents directed against topoisomerase I (irinotecan/CPT-11) and II (etoposide and doxorubicin), in vivo studies were performed in mice bearing the EMT-6 mammary tumor to assess efficacy, host tolerance and the resultant biochemical changes in topoisomerase mRNA and protein. Results: At 24 h after therapy, depletion of the target topoisomerase mRNA and protein with reciprocal increases in the alternate topoisomerase mRNA and, to a lesser extent, protein were noted. No therapeutic antagonism was found with any combination or sequence of agents, and therapeutic antagonism was noted with concurrent irinotecan/etoposide and sequential doxorubicin/irinotecan. Depletion of target topoisomerases by combined therapy beyond a threshold necessary for therapeutic efficacy produced no additional benefit. Conclusions: Antineoplastic therapy with combinations of topoisomerase I and II agents is feasible and may produce therapeutic synergy. The appropriate sequence may depend on the particular agents used. The rationale for such therapy, that topoisomerases I and II may have reciprocal and compensatory interactions, is supported by the biochemical data.

Reversal of in vivo drug resistance by the transforming growth factor-β inhibitor decorin

Transforming growth factor‐β (TGF‐β) has been implicated in the in vivo resistance of the EMT‐6/CTX and EMT‐6/CDDP murine mammary tumors. Both of these tumors have a higher number of intratumoral vessels than the EMT‐6/parent tumor. Animals bearing the resistant tumors have higher plasma levels of TGF‐β than animals bearing the parent tumors; however, upon treatment with cytotoxic therapies there is a greater rise in plasma TGF‐β levels in animals bearing the parent tumor than in animals bearing the resistant tumor. In situ hybridization for TGF‐β mRNA and immunohistochemical staining for TGF‐β protein showed that the resistant tumor levels of this growth factor are higher than those of the parent tumor prior to treatment; however, after cytotoxic therapy the increase in TGF‐β is greater in the parent tumor than in the resistant tumors. Treatment of tumor‐bearing animals with the naturally occurring TGF‐β inhibitor decorin did not alter the sensitivity of the parent tumor to cyclophosphamide or to CDDP as determined by tumor cell survival assay. However, administration of decorin increased the sensitivity of the EMT‐6/CTX tumor to cyclophosphamide and of the EMT‐6/CDDP tumor to CDDP so that the drug resistance of these tumors was nearly ablated. A similar pattern was observed in the drug response of the bone marrow granulocyte‐macrophage colony‐stimulating factor of animals bearing each of the 3 tumors. Int. J. Cancer, 71:49–58, 1997.

Transforming growth factor-β in in vivo resistance

Abstract The potential role of transforming growth factor-β in in vivo resistance was examined by administration of transforming growth factor-β-neutralizing antibodies to animals bearing the EMT-6/Parent tumor or the antitumor alkylating resistant tumors, EMT-6/CTX or EMT-6/CDDP. Treatment of tumor-bearing animals with anti-TGF-β antibodies by intraperitoneal injection daily on days 0–8 post-tumor cell implantation increased the sensitivity of the EMT-6/Parent tumor to cyclophosphamide (CTX) and cisplatin (CDDP) and markedly increased the sensitivity of the EMT-6/CTX tumor to CTX and the EMT-6/CDDP tumor to CDDP, as determined by tumor cell survival assay. Bone marrow granulocyte-macrophage colony-forming units (CFU-GM) survival was determined from these same animals. The increase in the sensitivity in the tumors upon treatment with the anti-TGF-β antibodies was also observed in increased sensitivity of the bone marrow CFU-GM to CTX and CDDP. Treatment of non-tumor-bearing animals with the anti-TGF-β regimen did not alter blood ATP or serum glucose level but did decrease serum lactate levels. This treatment also decreased hepatic glutathione, glutathione S-transferase, glutathione reductase, and glutathione peroxidase in non-tumor-bearing animals by 40–60% but increased hepatic cytochrome P450 reductase in these normal animals. Animals bearing the EMT-6/CTX and EMT-6/CDDP tumors had higher serum lactate levels than normal or EMT-6/Parent tumor-bearing animals; these were decreased by the anti-TGF-β regimen. Treatment of animals bearing any of the three tumors with the anti-TGF-β regimen decreased by 30–50% the activity of hepatic glutathione S-transferase and glutathione peroxidase, and increased by 35–80% the activity of hepatic cytochrome P450 reductase. In conclusion, treatment with transforming growth factor-β-neutralizing antibodies restored drug sensitivity in the alkylating agent-resistant tumors, altering both the tumor and host metabolic states.

In vivo modulation of several anticancer agents by β-carotene

The ability of the collagenase inhibitor minocycline and of β-carotene to act as positive modulators of cytotoxic anticancer agents was assessed in vitro and in vivo. Cell-culture studies were conducted using the human SCC-25 squamous carcinoma cell line. Simultaneous exposure of the cells to minocycline and β-carotene or 13-cis-retinoic acid along with cisplatin (CDDP) resulted in a small decrease in the cytotoxicity of the CDDP. The addition of each of the modulator combinations for 1 h or 24 h to treatment with melphalan (L-PAM) or carmustine (BCNU) resulted in greater-than-additive cytotoxicity with each of four regimens. The modulator combinations of minocycline and β-carotene applied for 1 h or 24 h and the modulator combination of munocycline and 13-cis-retinoic acid produced greater-than-additive cytotoxicity at 50 μM 4-hydroperoxycyclophosphamide (4-HC), whereas minocycline and 13-cis-retinoic acid applied for 1 h was antagonistic with 4-HC and the other modulator treatments at low concentrations of 4-HC resulted in subadditive cytotoxicity. The effect of treatment with β-carotene alone and in combination with several different anticancer agents was examined in two murine solid tumors, the FSaII fibrosarcoma and the SCC VII carcinoma. Administration of the modulators alone or in combination did not alter the growth of either tumor. Whereas increases in tumor growth delay occurred with the antitumor alkylating agents and β-carotene and with minocycline and β-carotene, a diminution in tumor growth delay was produced by 5-fluorouracil in the presence of these modulators. The modulator combination also resulted in increased tumor growth delay with Adriamycin and etoposide. Tumor-cell survival assay showed increased killing of FSaII tumor cells with the modulator combination and melphalan or cyclophosphamide as compared with the drugs alone. These results indicate that further investigation of this modulator strategy is warranted.