组蛋白去乙酰化酶抑制剂LBH589体外诱导人多发性骨髓瘤细胞株U266凋亡及其机制

目的研究新一代组蛋白去乙酰化酶（HDAC）抑制剂LBH589单药或联合中药禹州漏芦含药血清对人多发性骨髓瘤（MM）细胞株U266诱导凋亡作用及其机制。方法利用免疫印迹技术（Westernblot）分析不同浓度LBH589作用HDAC6特异底物α-微管蛋白乙酰化水平;采用免疫沉淀（IP）法检测不同浓度LBH589作用后热休克蛋白90（HSP90）与其客户蛋白的亲和力。结果Westernblot分析显示不同浓度LBH589（0、20、50nmol／L）单药及50nmol／L与禹州漏芦含药血清（1g／m1）联合均能够抑制U266细胞增殖,随着药物浓度的增加和作用时间的延长,仪。微管蛋白乙酰化水平、HSP90乙酰化的程度逐渐上调,变化呈剂量依赖性（P〈0．05）,且LBH589与禹州漏芦含药血清联合组抑制作用较单药组明显（均P〈0．05）。结论LBH589能够抑制MM细胞增殖,阻滞细胞周期,诱导人MM细胞株U266凋亡。     

Histone deacetylase inhibitor LBH589 reactivates silenced estrogen receptor alpha (ER) gene expression without loss of DNA hypermethylation

Our previous studies demonstrated that inhibition of histone deacetylases (HDACs) by trichostatin A reactivates estrogen receptor alpha (ER) gene expression in ER-negative breast cancer cells. Here, we use the clinically relevant HDAC inhibitor, LBH589 (LBH) to explore the roles of HDAC in ER gene silencing. In the ER-negative human breast cancer lines, MDA-MB-231 and MDA-MB-435, treatment with LBH for 24 hours restored ER mRNA and protein expression without a concomitant demethylation of the CpG island at the ER promoter. The expression of ER mRNA was sustained at least 96 hours after withdrawal of LBH treatment. Restoration of ER expression by LBH enhanced 4-hydroxy-tamoxifen sensitivity in MDA-MB-231 cells. The molecular mechanisms by which LBH reactivated silenced ER gene in MDA-MB-231 cells were examined with emphasis on chromatin structure reorganization. By chromatin immunoprecipitation analysis, LBH treatment released DNMT1, HDAC1, and the H3 lysine 9 (H3-K9) methyltransferase SUV39H 1 from the ER promoter. Such changes were associated with an active chromatin formation manifested as accumulation of acetylated histones H3 and H4, a decrease in methylated H3-K9, and an impaired binding of heterochromatin protein 1 (HP1 alpha) at the promoter. Our findings suggest that HDAC inhibitors could restore expression of the silenced ER gene by reorganizing the heterochromatin-associated proteins without alteration in promoter DNA hypermethylation.     

Cytotoxic activity of the histone deacetylase inhibitor panobinostat (LBH589) in anaplastic thyroid cancer in vitro and in vivo

Anaplastic thyroid carcinoma (ATC) has a rapidly fatal clinical course, being resistant to multimodal treatments. Microtubules, α/β tubulin heterodimers, are crucial in cell signaling, division and mitosis and are among the most successful targets for anticancer therapy. Panobinostat (LBH589) is a potent deacetylase inhibitor acting both on histones and nonhistonic proteins, including α-tubulin. In vitro LBH589, evaluated in three ATC cell lines (BHT-101, CAL-62 and 8305C), resulted in impairment of cell viability, inhibition of colony formation, cell cycle arrest and apoptosis induction. Mechanistically, we showed that LBH589 not only affected the expression of p21 and cyclin D1, but markedly determined microtubule stabilization as evidenced by tubulin acetylation and increased tubulin polymerization. In a SCID xenograft model implanted with CAL-62 cells, the cytotoxic properties of LBH589 were confirmed. The drug at the dose of 20 mg/kg significantly impaired tumor growth (final tumor volume 2.5-fold smaller than in untreated animals); at this dose, no relevant side effects were observed. In tumors of treated animals, a significant reduction of Ki67, which was negatively correlated with tubulin acetylation, was observed. Moreover, acetyl-tubulin levels negatively correlated with tumor volume at sacrifice, reinforcing the opinion that tubulin acetylation has a role in the inhibition of tumor growth. In conclusion, LBH589, acting on both histones and nonhistonic proteins in anaplastic thyroid cancer, appears to be a promising therapeutic agent for the treatment of this kind of cancer which is known not to respond to conventional therapy.     

Inhibition of the proliferation of acquired aromatase inhibitor-resistant breast cancer cells by histone deacetylase inhibitor LBH589 (panobinostat)

Aromatase inhibitors (AIs) are important drugs for treating postmenopausal patients with hormone receptor-positive breast cancer. However, acquired resistance to AI therapies is a significant problem. Our study has revealed that the histone deacetylase inhibitor LBH589 treatment abrogated growth of AI-resistant cells in vitro and in vivo, causing cell cycle G2/M arrest and induced apoptosis. LBH589 treatment also reduced the level of NF-κB1 which is overexpressed when AI resistance develops. Analyzing paired tumor specimens from 12 patients, we found that NF-κB1 expression was increased in recurrent AI-resistant tumors as compared to the paired primary tumors before AI treatment. This finding was consistent with up-regulated NF-κB1 expression seen in a collection of well-established AI-resistant cell lines. Furthermore, knockdown of NF-κB1 expression significantly suppressed the proliferation of AI-resistant cells. Treatment of AI-resistant cell lines with LBH589 suppressed NF-κB1 mRNA and protein expression. In addition, LBH589 treatment abrogated growth of AI-resistant tumors in mice, and was associated with significantly decreased levels of NF-κB1 in tumors. In all, our findings strongly support further investigation of LBH589 as a novel therapeutic strategy for patients with AI-resistant breast cancer, in part by suppressing the NF-κB1 pathway.     

Progress in clinical trial of histone deacetylase (HDAC) inhibitors for non-small cell lung cancers

Histone deacetylase (HDAC) inhibitors, which represent a structural y diverse group of molecules, have emerged as a novel therapeutic class of molecules with significant anticancer potential. Vorinostat and romidepsin, known as the first generation of HDAC inhibitors, were approved in the United States for the treatment of T-celllymphomas. Preliminary activity of HDAC inhibitors has also been observed in non-smal celllung cancer (NSCLC) in combination with the existing treatment regimens, of which is the focus of the current review.     

Enhanced radiation-induced cell killing and prolongation of gammaH2AX foci expression by the histone deacetylase inhibitor MS-275

Histone deacetylase (HDAC) inhibitors are undergoing clinical evaluation for cancer therapy. Because HDAC modulates chromatin structure and gene expression, parameters considered to influence radioresponse, we have investigated the effects of the HDAC inhibitor MS-275 on the radiosensitivity of two human tumor cell lines (DU145 prostate carcinoma and U251 glioma). Acetylation status of histones H3 and H4 was determined as a function of time after MS-275 addition to and removal from culture medium. Histone acetylation increased by 6 h after MS-275 addition, reaching a maximum between 24 and 48 h of exposure; providing fresh drug-free medium then resulted in a decrease in histone acetylation that began by 6 h and approached untreated levels by 16 h. Treatment of cells with MS-275 for 48 h followed by irradiation had little or no effect on radiation-induced cell death. However, exposure to MS-275 before and after irradiation resulted in an increase in radiosensitivity with dose enhancement factors of 1.9 and 1.3 for DU145 and U251 cells, respectively. This MS-275 treatment protocol did not result in a redistribution of the cells into a more radiosensitive phase of the cell cycle or in an increase in apoptosis. However, MS-275 did modify the time course of gammaH2AX expression in irradiated cells. Whereas there was no significant difference in radiation-induced gammaH2AX foci at 6 h, the number of cells expressing gammaH2AX foci was significantly greater in the MS-275-treated cells at 24 h after irradiation. These results indicate that MS-275 can enhance radiosensitivity and suggest that this effect may involve an inhibition of DNA repair.     

Histone deacetylase inhibitor, trichostatin A, increases the chemosensitivity of anticancer drugs in gastric cancer cell lines

Epigenetic alterations of the histone acetylation play an important role in the regulation of gene expression associated with cell cycles and apoptosis that may affect the chemosensitivity of gastric carcinomas. Recently, a histone deacetylase inhibitor, trichostatin A (TSA), was proven to be a chemo-sensitizer on human erythroleukemia cells. With the aim of improving the chemotherapeutic efficacy of gastric carcinoma, the effect of TSA on the chemosensitivity of several anticancer drugs in gastric carcinoma cells was investigated. Human gastric cancer cell lines, OCUM-8 and MKN-74, and 5 anticancer drugs, 5-fluorouracil (5-FU), paclitaxel (PTX), oxaliplatin (OXA), irinotecan (SN38) and gemcitabine (GEM) were used. In both gastric cancer cell lines, a synergistic anti-proliferative effect by the combination of TSA (30 ng/ml) with 5-FU, PTX or SN38 showed a synergistic anti-proliferative effect in OCUM-8 and MKN-74 cells. TSA increases the expression of p21, p53, DAPK-1 and the DAPK-2 gene in both OCUM-8 and MKN-74 cells. In conclusion, TSA is a promising chemotherapeutical agent in combination with anticancer drugs of 5-FU, PTX and SN38 in gastric cancer cell lines. The up-regulation of p53, p21, DAPK-1 and DAPK-2 might be associated with the synergistic effect of TSA.     

Histone modification in the TGFbetaRII gene promoter and its significance for responsiveness to HDAC inhibitor in lung cancer cell lines

We previously reported silencing of the TGF-beta type II receptor gene (TGFbetaRII), involving histone deacetylation, instead of DNA methylation (DNA-Me). Because different histone modifications may play crucial roles in the epigenetic alterations, we further studied links with silencing of the TGFbetaRII gene promoter in six lung cancer cell lines. ChIP assays demonstrated three chromatin patterns for this gene silencing (Pattern I: histone H3 acetylation (H3-Ac)(+/-)/histone H3 lysine 4 methylation (H3K4-Me)(+)/DNA-Me(-), Pattern II; H3-Ac(-)/H3K4-Me(+/-)/DNA-Me(-), and Pattern III; H3-Ac(-)/H3K4-Me(-)/DNA-Me(+)), indicating possible progressive alterations with H3K4-Me alteration. With exposure to a histone deacetylase inhibitor (HDAC-I), trichostatin A, cell lines with the pattern II demonstrated strong and persistent induction of TGFbetaRII expression, while those with the pattern III showed only weak or no induction. ACC-LC-91 cell line, one of the pattern II examples demonstrated strong and continuous induction of H3K4-Me similar to TGFbetaRII expression. In contrast, ACC-LC-176 with the pattern III showed only weak and transient induction of H3K4-Me, similar to TGFbetaRII expression. Treatment with 5-aza-2'-deoxycytidine (5aza-dC) in addition to HDAC-I resulted in strong and continuous induction of TGFbetaRII expression and H3K4-Me in ACC-LC-176, although 5aza-dC alone was without such effects. In ACC-LC-91, both H3-Ac and H3K4-Me were promptly and simultaneously induced by HDAC-I, and similarly inhibited by wortmannin, a PI3K family inhibitor, together with TGFbetaRII induction. These findings suggested progressive alterations of chromatin configuration including H3K4-Me alteration in TGFbetaRII gene silencing. A possible involvement of a wortmannin-sensitive kinase in histone modification was also suggested.     

Phase II trial of the histone deacetylase inhibitor pivaloyloxymethyl butyrate (Pivanex, AN-9) in advanced non-small cell lung cancer

This multicenter phase II trial evaluated the therapeutic activity and safety profile of pivaloyloxymethyl butyrate (Pivanex, AN-9) as a single agent in refractory non-small cell lung cancer (NSCLC). Pivanex (2.34 g/m2 per day) was administered as a 6-h continuous intravenous infusion, daily for 3 days, and repeated every 21 days until disease progression. Forty-seven patients were treated. More than 90% of patients had received both a platinum compound and a taxane and 32% had received three or more prior chemotherapy regimens. The most common toxicities were transient grade 1-2 fatigue (34%), nausea (17%), and dysgeusia (11%). Three patients had partial responses (6.4 and 95%; CI 1.4-18.7%) and 14 patients had stable disease for > or =12 weeks (30%). Median survival for all patients was 6.2 months with 1-year survival of 26%. For patients who received fewer than three prior chemotherapy regimens, median survival was 7.8 months and 1-year survival was 31%. Pivanex is well tolerated and appears to be active as a single agent in patients with advanced NSCLC refractory to previous chemotherapy. Based on its therapeutic activity and favorable safety profile, further studies of Pivanex in NSCLC, particularly in combination with current chemotherapeutic agents, are warranted.     

Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines

Programmed cell death (PCD) is a genetically regulated pathway that is altered in many cancers. This process is, in part, regulated by the ratio of PCD inducers (Bax) or inhibitors (Bcl-2). An abnormally high ratio of Bcl-2 to Bax prevents PCD, thus contributing to resistance to chemotherapeutic agents, many of which are capable of inducing PCD. Non-small cell lung cancer (NSCLC) cells demonstrate resistance to these PCD-inducing agents. If Bcl-2 prevents NSCLC cells from entering the PCD pathway, then reducing the amount of endogenous Bcl-2 product may allow these cells to spontaneously enter the PCD pathway. Our purpose was to determine the effects of bcl-2 antisense treatment on the levels of programmed cell death in NSCLC cells. First, we determined whether bcl-2 and bax mRNA were expressed in three morphologically distinct NSCLC cell lines: NCI-H226 (squamous), NCI-H358 (adenocarcinoma), and NCI-H596 (adenosquamous). Cells were then exposed to synthetic antisense bcl-2 oligonucleotide treatment, after which programmed cell death was determined, as evidenced by DNA fragmentation. Bcl-2 protein expression was detected immunohistochemically. All three NSCLC cell lines expressed both bcl-2 and bax mRNA and had functional PCD pathways. Synthetic antisense bcl-2 oligonucleotide treatment resulted in decreased Bcl-2 levels, reduced cell proliferation, decreased cell viability, and increased levels of spontaneous PCD. This represents the first evidence that decreasing Bcl-2 in three morphologically distinct NSCLC cell lines allows the cells to spontaneously enter a PCD pathway. It also indicates the potential therapeutic use of antisense bcl-2 in the treatment of NSCLC.     

In vitro study of CI-994, a histone deacetylase inhibitor, in non-small cell lung cancer cell lines

CI-994 (N-acetyldinaline) is a novel oral compound with a wide spectrum of antitumor activity in preclinical models, in vitro and in vivo. The mechanism of action may involve inhibition of histone deacetylation and cell cycle arrest. We studied the action of CI-994 on two non-small cell lung cancer (NSCLC) cell lines: A-549 (adenocarcinoma) and LX-1 (squamous cell carcinoma). Different drug concentrations were tested, ranging from 0.01 to 160 microM at 24, 48, and 72 h of treatment, with MTT assay. A concentration-dependent cell survival inhibition was observed, with an IC50 at 80 microM. The effect of CI-994, as demonstrated by recovery experiments, was cytostatic and seemed to be superimposable in both cell lines. Cytofluorimetric analysis to assess cell cycle perturbation and apoptosis was performed after 24 h of treatment, indicating a cell block with concomitant increase at G0/G1 phase, a reduction at S phase level at 20, 40, 80, and 160 microM, and apoptosis at the higher concentration (160 microM). When CI-994 was combined with antineoplastic agents commonly used in NSCLC management, a marked synergism of action (R = 1.8, R = 1.5) was observed between CI-994 (40 microM) and gemcitabine (0.01 microM) at 48 and 72 h of treatment. The same result was obtained with docetaxel (0.001 microM) combination (R = 1.4, R = 1.2), but no synergism of action was noted with paclitaxel. CI-994 showed no radiopotentiating effects, when combined with 100, 200, or 400 cGy irradiation. In conclusion, our experiments indicate that CI-994 is a promising novel cytostatic for the treatment of NSCLC. Its use in combination with standard anticancer agents, such as gemcitabine and docetaxel, is warranted.     

Gemcitabine (Gemzar) in non-small cell lung cancer

Of the new chemotherapeutic substances of the last decade, gemcitabine (Gemzar, Eli Lilly) is probably the most valuable for the treatment of early and advanced stage non-small cell lung cancer (NSCLC). When used as a single agent in both chemotherapeutically pretreated and chemotherapy-naive patients, gemcitabine shows an objective tumor regression rate of approximately 20%. Gemcitabine's unique mechanism of action and its lack of overlapping toxicity with other cytotoxic agents also define it as an ideal candidate for combination therapy. Early clinical development has included single-agent first- and second-line treatment, doublet combination regimens and incorporation into multimodality treatment strategies for operable and inoperable locally advanced nonmetastatic NSCLC. Gemcitabine/platinum-based combination chemotherapy has become the most attractive treatment standard for NSCLC patients in good clinical condition. The role of gemcitabine in the concurrent or sequential application of chemo- and radiotherapy for inoperable locally advanced NSCLC has also been addressed in several Phase I and II studies. Based on data available, gemcitabine can be safely administered in combination with radiotherapy. This review summarizes results from representative Phase I, II and III studies in order to underline gemcitabine's clinical importance for patients suffering from early and advanced NSCLC.     

Dichloroacetic acid (DCA) synergizes with the SIRT2 inhibitor Sirtinol and AGK2 to enhance anti-tumor efficacy in non-small cell lung cancer

Combination chemotherapy is a potentially promising approach to enhance anticancer activity, overcome drug resistance, and improve disease-free and overall survival. The current study investigates the antitumor activity of sodium dichloroacetic acid (DCA) in combination with SIRT2 inhibitor Sirtinol and AGK2. We found that combining DCA with Sirtinol produced a synergistic therapeutic benefit in A549 and H1299 NSCLC cells in vitro and in a mouse A549 xenograft model. Synergistic potentiation of oxidative phosphorylation (OXPHOS) was observed, including decreased glucose consumption, decreased lactate production, increased OCR and increased ROS generation, possibly via co-targeting pyruvate dehydrogenase alpha 1(PDHA1). Mechanically, AGK2 and Sirtinol were found to increase the lysine-acetylation and decrease the serine-phosphorylation of PDHA1, which enabled the two inhibitors to synergize with DCA to further activate PDHA1. Besides, a AMPKα-ROS feed-forward loop was notably activated after the combined treatments compared with mono-therapy. Our results indicate that the combination of DCA and SIRT2 inhibitor may provide a promising therapeutic strategy to effectively kill cancer cells.     

HDAC6-mediated EGFR stabilization and activation restrict cell response to sorafenib in non-small cell lung cancer cells

Sorafenib is a multi-targeted kinase inhibitor and has been the subject of extensive clinical research in advanced non-small cell lung cancer (NSCLC). However, sorafenib fails to improve overall survival of patients with advanced NSCLC. The molecular mechanisms that account for this phenomenon are unclear. Here we show that sorafenib treatment stabilizes epidermal growth factor receptor (EGFR) and activates EGFR pathway. Moreover, this is partly mediated by stabilization of histone deacetylase 6 (HDAC6), which has been shown to regulate EGFR endocytic trafficking and degradation. Overexpression of HDAC6 confers resistance to sorafenib in NSCLC cells. Inhibition of HDAC6 with selective inhibitors synergizes with sorafenib to kill NSCLC cells via inhibition of sorafenib-mediated EGFR pathway activation. Taken together, our findings might partly explain the failure of Phase III trial of sorafenib in improving overall survival of advanced NSCLC patients and bear possible implications for the improvement on the efficacy of sorafenib in treatment of NSCLC.     

ZD1839 (Iressa) in non-small cell lung cancer

Despite the advent of cisplatin-based combination chemotherapy for advanced non-small cell lung cancer (NSCLC), the prognosis for this patient population remains poor. Novel biologically targeted agents currently in development have the potential for greater efficacy against NSCLC, and possibly less toxicity than is associated with conventional cytotoxic chemotherapy. The epidermal growth factor receptor (EGFR) is recognized as a potentially useful target, and the small molecule, orally active EGFR-tyrosine kinase inhibitor ZD1839 (Iressa) is currently the furthest along in clinical development of the anti-EGFR agents. This review summarizes the currently available clinical data on the use of ZD1839 in the treatment of NSCLC.     

Gefitinib (Iressa) trials in non-small cell lung cancer

Gefitinib (Iressa) is a synthetic anilinoquinazoline capable of inhibiting the epidermal growth factor receptor tyrosine kinase in vitro at nanomolar concentrations. In phase I trials, gefitinib was well tolerated at doses above that required to induce antitumor effects in vitro. Notably, antitumor activity was observed in lung cancer patients. These findings resulted in the initiation of phase II trials employing gefitinib monotherapy in patients with recurrent non-small cell lung cancer (the so-called IDEAL trials). Study participants were randomized to 250 mg or 500 mg of gefitinib per day. Objective response rates between 10 and 20% were achieved with minimal host related toxicity (mainly acne like rash and mild diarrhea). Median survivals ranged between 6 and 8 months. Subsequently, phase III trials (the so-called INTACT trials) combined gefitinib and chemotherapy in chemonaive patients with advanced non-small cell lung cancer. These trials failed to demonstrate a survival advantage with the addition of gefitinib to standard platinum-based chemotherapy regimens. However, overall host related toxicities were not substantially worsened with the addition of gefitinib to chemotherapy. Further studies employing single agent gefitinib as well as regimens employing a different sequencing of chemotherapy and gefitinib are planned in recurrent and previously untreated lung cancer patients.