Combination Of Suberoylanilide Hydroxamic Acid Research Articles

Improving therapeutic potential in breast cancer via histone deacetylase inhibitor loaded nanofibrils.

Epigenetic modifications play a significant role in cancer progression, making them potential targets for therapy. Histone deacetylase inhibitors have shown promise in inhibiting cancer cell growth, including in breast cancer (BC). In this research, we examined the potential of using suberoyl anilide hydroxamic acid (SAHA)-loaded β-lg nanofibrils as a drug delivery system for triple-negative BC cell lines. We assessed their impact on cell cycle progression, apoptosis, levels of reactive oxygen species, and mitochondrial membrane potential in cancer cells. The combination of SAHA and β-lg nanofibrils demonstrated enhanced efficacy in inhibiting cell growth, inducing cell cycle arrest, and promoting apoptosis (43.78%) compared to SAHA alone (40.09%). Moreover, it effectively targeted cancer cells without promoting drug resistance while using a low concentration of the nanofibrils. These findings underscore the promising potential of nanofibril-based drug delivery systems for BC treatment.

KLF5 Promotes Tumor Progression and Parp Inhibitor Resistance in Ovarian Cancer.

One major characteristic of tumor cells is the aberrant activation of epigenetic regulatory elements, which remodelthe tumor transcriptome and ultimately promotecancer progression and drug resistance. However, the oncogenic functions and mechanisms of ovarian cancer (OC) remain elusive. Here, super-enhancer (SE) regulatory elements that are aberrantly activated in OC are identified and it is found that SEs drive the relative specific expression of the transcription factor KLF5 in OC patients and poly(ADP-ribose) polymerase inhibitor (PARPi)-resistant patients. KLF5 expression is associated with poor outcomes in OC patients and can drive tumor progression in vitro and in vivo. Mechanistically, KLF5 forms a transcriptional complex with EHF and ELF3 and binds to the promoter region of RAD51 to enhance its transcription, strengthening the homologous recombination repair (HRR) pathway. Notably, the combination of suberoylanilide hydroxamic acid (SAHA) and olaparib significantly inhibits tumor growth and metastasis of PARPi-resistant OC cells with high KLF5. In conclusion, it is discovered that SEs-driven KLF5 is a key regulatory factor in OC progression and PARPi resistance;and potential therapeutic strategies for OC patients with PARPi resistance and high KLF5 are identified.

Pharmacological Inhibitory Effect of Imetelstat, a Novel Human Telomerase Inhibitor in Diffuse Large B-Cell Lymphoma and Peripheral T-Cell Lymphoma

Telomerase activity is present in some non-Hodgkin lymphomas (NHL), including diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell lymphomas (PTCL), and correlated with proliferation index, suggesting anti-telomerase drugs may be efficacious in some types of NHL (Ely et al. 2000). Imetelstat, a telomerase inhibitor that specifically targets the RNA template of human telomerase, is a potent, first-in-class, competitive inhibitor of telomerase enzymatic activity and has demonstrated clinical activity in myeloid malignancies (Steensma et al. 2021; Mascarenhas et al. 2021). No studies have been conducted to demonstrate the effects of imetelstat in DLBCL and PTCL. In our study, we characterized telomerase activity and telomere length, as well as the in-vitro effects of imetelstat on cell viability and apoptosis of a panel of DLBCL and PTCL cell lines. Six DLBCL cell lines, including germinal B cells (GCB: DOHH2, HT, SUDHL4), non-germinal B cells (HBL-1, TMD8), and one high-grade B cell lymphoma line RC with MYC and BCL2 gene rearrangements (double hit lymphomas; ), and five PTCL cell lines (Karpas 299, H9, MAC2A, SUDHL1, and SUPM2) were tested for imetelstat single agent activity as assessed by change of telomerase activity using ScienCell's Telomerase Activity Quantification qPCR Assay Kit, cell viability by Cell Titer-Glo Luminescent Cell Viability Assay and apoptosis by Annexin V/Propidium Iodide flow assay. Baseline telomerase activity and telomere length were measured for each cell line to correlate with imetelstat activity. In addition, PTCL cell lines were treated with Suberoylanilide hydroxamic acid (SAHA) and imetelstat to determine if the combination had an impact on cell viability over either of the single agents. All DLBCL and PTCL cell lines have higher baseline telomerase activity with Ct 20.6-23.2 cycles than the telomerase-positive cell lysate control with Ct 30.6 cycles used in the assay. The dose and time-dependent effects of imetelstat were examined through cell viability and apoptosis assays. Imetelstat sensitivity was higher in DLBCL than in PTCL cell lines used in this study. Preliminary experiments determined that IC50 3-17.5 μM imetelstat markedly reduced cell viability and induced apoptosis in DLBCL cell lines. Specifically, non-GCB DLBCL cell lines (IC50 = 3-4.1 μM) were more sensitive than the GCB-DLBCL (IC50 = 6-8.2 μM). While the level of effects in cell viability and apoptosis by imetelstat varied in the DLBCL cell lines, the double hit DLBCL cell line RC was the least sensitive to imetelstat (IC50 = 17.5 μM) after treatment for 8 days. In contrast, compared to DLBCL cell lines, imetelstat single-agent activity on cell viability was limited in PTCL cell lines, although a time and dose-dependent reduction of telomerase activity were noted. Additionally, the greater inhibitory effect in DLBCL than in PTCL cell lines by imetelstat treatment may be attributed to higher telomerase activity observed in DLBCL compared to PTCL cell lines. Furthermore, the PTCL cell lines had a ~7.3 fold longer telomere length than DLBCL cell lines, which was associated with less response to imetelstat than DLBCL lines. A combination of SAHA with imetelstat was more effective than a single agent alone, synergistic activity was observed in Karpas 299, while other PTCL lines had an additive effect. Our findings indicate that imetelstat was more sensitive in non-GCB subtypes than in GCB-DLBCL. While in PTCL cell lines, the activity of imetelstat was enhanced when combined with SAHA. In the lymphoma landscape, telomerase may be a therapeutic target for ABC-type DLBCL; the novel telomerase inhibitor Imetelstat may present one of the potential treatment options.

HDAC AND NFKB INHIBITORS REDUCE CANCER STEM CELLS AND SENSITIZE MUCOEPIDERMOID CARCINOMA CELL LINES TO CISPLATIN

Objectives To evaluate the potential of histone deacetylase (HDAC) suberoylanilide hydroxamic acid (SAHA) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) (emetine) inhibitors, isolated and associated with cisplatin (CDDP) in salivary gland mucoepidermoid carcinoma (MEC) cell lines, specifically through cancer stem cell (CSC) modulation. Study Design Cell viability was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Immunofluorescence staining was performed to identify the acetylation of lysine 9 from histone H3 protein (H3k9ac) and phosphorylated protein p65 (NFkB pathway). CSC was evaluated by quantifying the enzymatic activity of aldehyde dehydrogenases (ALDH) using flow cytometry and by in vitro tumorsphere formation. Results A single administration of SAHA (1 μM) induced histone acetylation, whereas 0.05 μM of emetine resulted in inhibition of the NF-kB pathway. We showed a disruption of CSC, illustrated by a reduction of enzymatic activity of ALDH and inhibition of tumorsphere formation. Furthermore, we observed that MEC tumor cells were sensitized to CDDP after the administration of a combination of SAHA and emetine. Conclusions Our findings suggest that HDAC and NF-kB inhibitors might be an alternative approach to treat MEC through disruption of CSC and sensitizing cells to conventional therapy. To evaluate the potential of histone deacetylase (HDAC) suberoylanilide hydroxamic acid (SAHA) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) (emetine) inhibitors, isolated and associated with cisplatin (CDDP) in salivary gland mucoepidermoid carcinoma (MEC) cell lines, specifically through cancer stem cell (CSC) modulation. Cell viability was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Immunofluorescence staining was performed to identify the acetylation of lysine 9 from histone H3 protein (H3k9ac) and phosphorylated protein p65 (NFkB pathway). CSC was evaluated by quantifying the enzymatic activity of aldehyde dehydrogenases (ALDH) using flow cytometry and by in vitro tumorsphere formation. A single administration of SAHA (1 μM) induced histone acetylation, whereas 0.05 μM of emetine resulted in inhibition of the NF-kB pathway. We showed a disruption of CSC, illustrated by a reduction of enzymatic activity of ALDH and inhibition of tumorsphere formation. Furthermore, we observed that MEC tumor cells were sensitized to CDDP after the administration of a combination of SAHA and emetine. Our findings suggest that HDAC and NF-kB inhibitors might be an alternative approach to treat MEC through disruption of CSC and sensitizing cells to conventional therapy.

Regulation of Opsin Gene Expression by DNA Methylation and Histone Acetylation.

One important role of epigenetic regulation is controlling gene expression in development and homeostasis. However, little is known about epigenetics’ role in regulating opsin expression. Cell cultures (HEK 293, Y79, and WERI) producing different levels of opsins were treated with 5-aza-2’-deoxycytidine (5-Aza-dc) and/or sodium butyrate (SB) or suberoylanilide hydroxamic acid (SAHA) for 72 h. Global DNA methylation, site-specific methylation, and expressions of opsins were measured by LUMA assay, bisulfite pyrosequencing, and qPCR, respectively. Mouse retinal explants from wild-type P0/P1 pups were ex vivo cultured with/without 5-Aza-dc or SAHA for 6 days. The morphology of explants, DNA methylation, and expressions of opsins was examined. The drugs induced global DNA hypomethylation or increased histone acetylation in cells, including DNA hypomethylation of rhodopsin (RHO) and L-opsin (OPN1LW) and a concomitant increase in their expression. Further upregulation of RHO and/or OPN1LW in HEK 293 or WERI cells was observed with 5-Aza-dc and either SB or SAHA combination treatment. Mouse retinal explants developed normally but had drug-dependent differential DNA methylation and expression patterns of opsins. DNA methylation and histone acetylation directly regulate opsin expression both in vitro and ex vivo. The ability to manipulate opsin expression using epigenetic modifiers enables further study into the role of epigenetics in eye development and disease.

Suberoylanilide hydroxamic acid overcomes erlotinib-acquired resistance via phosphatase and tensin homolog deleted on chromosome 10-mediated apoptosis in non-small cell lung cancer.

Background:Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, are widely used to treat non-small cell lung cancer (NSCLC). However, acquired resistance is unavoidable, impairing the anti-tumor effects of EGFR-TKIs. It is reported that histone deacetylase (HDAC) inhibitors could enhance the anti-tumor effects of other antineoplastic agents and radiotherapy. However, whether the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) can overcome erlotinib-acquired resistance is not fully clear.Methods:An erlotinib-resistant PC-9/ER cell line was established through cell maintenance in a series of erlotinib-containing cultures. NSCLC cells were co-cultured with SAHA, erlotinib, or their combination, and then the viability of cells was measured by the 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and apoptosis was determined by flow cytometry and western blotting. Finally, the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was assessed by western blotting.Results:The half-maximal inhibitory concentration of parental PC-9 cells was significantly lower than the established erlotinib-acquired resistant PC-9/ER cell line. PC-9/ER cells demonstrated reduced expression of PTEN compared with PC-9 and H1975 cells, and the combination of SAHA and erlotinib significantly inhibited cell growth and increased apoptosis in both PC-9/ER and H1975 cells. Furthermore, treating PC-9/ER cells with SAHA or SAHA combined with erlotinib significantly upregulated the expression of PTEN mRNA and protein compared with erlotinib treatment alone.Conclusions:PTEN deletion is closely related to acquired resistance to EGFR-TKIs, and treatment with the combination of SAHA and erlotinib showed a greater inhibitory effect on NSCLC cells than single-drug therapy. SAHA enhances the suppressive effects of erlotinib in lung cancer cells, increasing cellular apoptosis and PTEN expression. SAHA can be a potential adjuvant to erlotinib treatment, and thus, can improve the efficacy of NSCLC therapy.

A potentiated cooperation of carbonic anhydrase IX and histone deacetylase inhibitors against cancer

The emergence of tumour recurrence and resistance limits the survival rate for most tumour-bearing patients. Only, combination therapies targeting pathways involved in the induction and in the maintenance of cancer growth and progression might potentially result in an enhanced therapeutic efficacy. Herein, we provided a prospective combination treatment that includes suberoylanilide hydroxamic acid (SAHA), a well-known inhibitor of histone deacetylases (HDACs), and SLC-0111, a novel inhibitor of carbonic anhydrase (CA) IX. We proved that HDAC inhibition with SAHA in combination with SLC-0111 affects cell viability and colony forming capability to greater extent than either treatment alone of breast, colorectal and melanoma cancer cells. At the molecular level, this therapeutic regimen resulted in a synergistically increase of histone H4 and p53 acetylation in all tested cell lines. Overall, our findings showed that SAHA and SLC-0111 can be regarded as very attractive combination providing a potential therapeutic strategy against different cancer models.

Adjuvant Epigenetic Therapy of Decitabine and Suberoylanilide Hydroxamic Acid Exerts Anti-Neoplastic Effects in Acute Myeloid Leukemia Cells.

Atypical epigenetic processes including histone acetylation and DNA methylation have been identified as a fundamental theme in hematologic malignancies. Such mechanisms modify gene expression and prompt, in part at least, the initiation and progression of several malignancies including acute myeloid leukemia. In the current study we determined the effects of treating KG-1 and U937 acute myeloid leukemia (AML) cells, in vitro, with the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), or with a DNMT inhibitor, decitabine (DAC), or their combination, on cell proliferation, cell cycle progression, apoptosis, and expression of apoptosis-related proteins. Each of SAHA and DAC attenuated cell proliferation and induced cell cycle arrest and apoptotic cell death of KG-1 and U937 cell lines. Besides, their sequential combination improved the obtained anti-neoplastic effect: significant augmentation of growth inhibition and apoptosis induction as compared to cells treated with either drug alone. This effect was featured by the upregulated expression of Bax, cytochrome c1, p21, and cleaved caspases 8, 9, and 3, signifying the activation of both the intrinsic and extrinsic pathways of apoptosis. The sequential combination of SAHA and DAC causes a profound antitumorigenic effect in AML cell lines by inducing the expression of tumor suppressor genes.

Synergistic Effect of Epigenetic Inhibitors Decitabine and Suberoylanilide Hydroxamic Acid on Colorectal Cancer In vitro.

Colorectal Cancer (CRC) is a common cause of oncological deaths worldwide. Alterations of the epigenetic landscape constitute a well-documented hallmark of CRC phenotype. The accumulation of aberrant DNA methylation and histone acetylation plays a major role in altering gene activity and driving tumor onset, progression and metastasis. In this study, we evaluated the effect of Suberoylanilide Hydroxamic Acid (SAHA), a panhistone deacetylase inhibitor, and Decitabine (DAC), a DNA methyltransferase inhibitor, either alone or in combination, on Caco-2 human colon cancer cell line in vitro. Our results showed that SAHA and DAC, separately, significantly decreased cell proliferation, induced apoptosis and cell cycle arrest of Caco-2 cell line. On the other hand, the sequential treatment of Caco-2 cells, first with DAC and then with SAHA, induced a synergistic anti-tumor effect with a significant enhancement of growth inhibition and apoptosis induction in Caco-2 cell line as compared to cells treated with either drug alone. Furthermore, the combination therapy upregulates protein expression levels of pro-apoptotic proteins Bax, p53 and cytochrome c, downregulates the expression of antiapoptotic Bcl-2 protein and increases the cleavage of procaspases 8 and 9; this suggests that the combination activates apoptosis via both the intrinsic and extrinsic pathways. Mechanistically, we demonstrated that the synergistic anti-neoplastic activity of combined SAHA and DAC involves an effect on PI3K/AKT and Wnt/β-catenin signaling. In conclusion, our results provide evidence for the profound anti-tumorigenic effect of sequentially combined SAHA and DAC in the CRC cell line and offer new insights into the corresponding underlined molecular mechanism.

Synergistic effect of epigenetic inhibitors decitabine and suberoylanilide hydroxamic acid on colorectal cancer in vitro

Colorectal cancer (CRC) is major public health burden and a common cause of oncological deaths worldwide. Alterations of the epigenetic landscape are a well‐documented hallmark of CRC phenotype. Accumulation of aberrant DNA methylation and histone acetylation orchestrates to modify gene activity and drive tumor onset, progression and metastasis. In this study we evaluated the effect of suberoylanilide hydroxamic acid (SAHA), a pan histone deacetylase inhibitor, and decitabine (DAC), a DNA methyltransferase inhibitor, either alone or in combination, on Caco‐2 human colon cancer cell line in vitro. Our results showed that SAHA and DAC, separately, significantly decreased cell proliferation, induced apoptosis and cell cycle arrest of Caco‐2 cell line. On the other hand, the sequential treatment of Caco‐2 cells, first with DAC and then with SAHA, induced a synergistic anti‐tumor effect; the sequential treatment significantly enhanced growth inhibition and apoptosis of Caco‐2 cell line in comparison to cells treated with either drug alone. Furthermore, the combination therapy upregulates protein expression levels of pro‐apoptotic proteins Bax, p53 and cytochrome c, down regulates the expression of anti‐apoptotic Bcl‐2 protein and increases the cleavage of procaspases 8 and 9; this suggests that the combination activates apoptosis via both the intrinsic and extrinsic pathways. Mechanistically, we demonstrated that the synergistic anti‐neoplastic activity of combined SAHA and DAC involves an effect on PI3K/AKT and Wnt/β‐catenin signaling pathways as it down regulates p‐PI3K, p‐AKT, Wnt1, p‐GSK3, cyclin A1+A2, cyclin E1 and up regulates p‐β‐catenin protein expression levels. In conclusion, our results provide evidence for the profound anti‐tumorigenic effect of sequentially combined SAHA and DAC in CRC cell line and offer new insights into the corresponding underlined molecular mechanism.Support or Funding InformationThis work was funded by SRDC, School of Arts and Sciences, Lebanese American UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The HDAC Inhibitor, SAHA, Combined with Cisplatin Synergistically Induces Apoptosis in Alpha-fetoprotein-producing Hepatoid Adenocarcinoma Cells.

Hepatoid adenocarcinoma (HAC) is a rare and aggressive gastrointestinal tract cancer that is characterized by hepatic differentiation and production of alpha-fetoprotein (AFP). Cisplatin is mainly used to treat HAC, but the efficacy is poor. Recently, the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), was approved as an anticancer agent. In this study, we investigated the anticancer effect of SAHA in combination with cisplatin in VAT-39 cells, a newly established HAC cell line. Cell viability and apoptosis were examined by MTT assay, flow cytometry and TUNEL assay. Expression of H3S10, cleaved caspase-3, Bax, and Bcl-2 were evaluated by immunohistochemistry and western blotting. AFP levels were examined in VAT-39 cells and culture medium. Combined treatment with cisplatin and SAHA efficiently inhibited cell proliferation and decreased cell viability. Apoptotic cells, but not necrotic cells, were significantly increased following the combined treatment, and an increase in the Bax/Bcl-2 ratio indicated that the combination of cisplatin and SAHA induced apoptosis through the mitochondrial pathway. VAT-39 cells treated with cisplatin and SAHA also partially lost their main characteristic of AFP production. We conclude that cisplatin and SAHA have a synergistic anticancer effect of inducing apoptosis, and that this combination treatment may be effective for HAC.

The combination of epigenetic drugs SAHA and HCI-2509 synergistically inhibits EWS-FLI1 and tumor growth in Ewing sarcoma.

PurposeEpigenetic regulation is crucial in mammalian development and maintenance of tissue-cell specific functions. Perturbation of epigenetic balance may lead to alterations in gene expression, resulting in cellular transformation and malignancy. Previous studies in Ewing sarcoma (ES) have shown that the Nucleosome Remodeling Deacetylase (NuRD) complex binds directly to EWS-FLI1 oncoprotein and modulates its transcriptional activity. The role of EWS-FLI1 as a driver of proliferation and transformation in ES is widely known, but the effect of epigenetic drugs on fusion activity remains poorly described. The present study evaluated the combination effects of the histone deacetylases inhibitor suberoylanilide hydroxamic acid (SAHA) and Lysine-specific demethylase1 inhibitor (HCI-2509) on different biological functions in ES and in comparison to monotherapy treatments.ResultsThe study of proliferation and cell viability showed a synergistic effect in most ES cell lines analyzed. An enhanced effect was also observed in the induction of apoptosis, together with accumulation of cells in G1 phase and a blockage of the migratory capacity of ES cell lines. Treatment, either in monotherapy or in combination, caused a significant decrease of EWS-FLI1 mRNA and protein levels and this effect is mediated in part by fusion gene promoter regulation. The anti-tumor effect of this combination was confirmed in patient-derived xenograft mouse models, in which only the combination treatment led to a statistically significant decrease in tumor volume.ConclusionsThe combination of SAHA and HCI-2509 is proposed as a novel treatment strategy for ES patients to inhibit the essential driver of this sarcoma and tumor growth.

Synergistic antitumor effect of suberoylanilide hydroxamic acid and cisplatin in osteosarcoma cells.

Cisplatin, as a first-line chemotherapy drug, has been widely applied for therapy of osteosarcoma. However, its application is limited by drug resistance and serious side effects, including nephrotoxicity and ototoxicity. Suberoylanilide hydroxamic acid (SAHA) is a newly developed histone deacetylase (HDAC) inhibitor, which is the first Food and Drug Administration-approved HDAC inhibitor for the treatment of cutaneous manifestations of T-cell lymphoma. However, SAHA as a monotherapy was revealed to be limited, particularly in solid tumors. In the present study, 143B osteosarcoma cells were treated with multiple concentrations of SAHA or cisplatin, either alone or combined. The morphological characteristics of the treated cells were observed using an inverted microscope. The cytotoxicity effects of the combination of SAHA and cisplatin on 143B cells were analyzed by MTT assay, colony formation assay, wound healing cell migration assay, cell apoptosis assay and cell cycle analysis. Western blot analysis was performed to detect the protein expression levels of B cell lymphoma-2 (Bcl-2)-associated X protein (Bax), Bcl-2, cleaved-caspase-3, cleaved-caspase-8 and cleaved-poly (ADP-ribose) polymerase (PARP). The experimental data indicated that the inhibition of cell proliferation in the combination group was significantly increased compared with that in single drug groups. Expression levels of pro-apoptotic protein were upregulated, whereas anti-apoptotic Bcl-2 was downregulated significantly in 143B cells following SAHA/cisplatin treatment. Taken together, the results revealed that the combination of SAHA and cisplatin inhibited the proliferation of 143B cells and induced their apoptosis synergistically, and this effectiveness may be mediated by caspase activation.

Abstract A67: The effects of SAHA and EGCG on metastatic potential in triple-negative breast cancer

Abstract Approximately 12% of breast cancers are triple negative, and are nearly two times more common in black women that white women. These cancers have a poorer short-term prognosis than other types due to the lack of targeted therapies. The purpose of this study was to use suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor used to treat cutaneous T cell lymphoma (CTCL), and epigallocatechin-3-gallate (EGCG), a DNA methyltransferase (DNMT) inhibitor found in green tea, to decrease metastatic potential in triple-negative breast cancer (TNBC) cells. SAHA and EGCG were administered singly and in combination to determine if the combination was more efficacious than either compound alone in their effects on cell viability and foci, or colony, formation. RNA and protein analysis was performed to evaluate epithelial versus mesenchymal markers, while scratch tests provided phenotypic evidence of changes in migration capability. We found that the combination of SAHA and EGCG at optimal concentrations of 3 μM SAHA and 5 μM reduced breast cancer cellular viability in an additive manner while this combination had no significant effect on control breast cells. This is a very low dose of EGCG compared to most anticancer studies. The colonies formed by the breast cancer cells decreased in size and number. Interestingly, EGCG administered singly increased colony formation. The combination of SAHA and EGCG also reactivated estrogen receptor alpha (ERα), and decreased the expression of miR-221/222, which are microRNAs involved in migration and invasion. The scratch tests supported these findings, with the breast cancer cells exhibiting a decreased ability to migrate. These findings demonstrate the abilities of combined SAHA and EGCG to decrease metastatic potential through epigenetic mechanisms in TNBC. Citation Format: Kayla Ashlyn Lewis, Trygve O. Tollefsbol. The effects of SAHA and EGCG on metastatic potential in triple-negative breast cancer [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A67.

SAHA and cisplatin sensitize gastric cancer cells to doxorubicin by induction of DNA damage, apoptosis and perturbation of AMPK-mTOR signalling

Chemotherapy remains the most prescribed anti-cancer therapy, despite patients suffering severe side effects and frequently developing chemoresistance. These complications can be partially overcome by combining different chemotherapeutic agents that target multiple biological pathways. However, selecting efficacious drug combinations remains challenging. We previously used fission yeast Schizosaccharomycespombe as a surrogate model to predict drug combinations, and showed that suberoylanilide hydroxamic acid (SAHA) and cisplatin can sensitise gastric adenocarcinoma cells toward the cytotoxic effects of doxorubicin. Yet, how this combination undermines cell viability is unknown. Here, we show that SAHA and doxorubicin markedly enhance the cleavage of two apoptosis markers, caspase 3 and poly-ADP ribose polymerase (PARP-1), and increase the phosphorylation of γH2AX, a marker of DNA damage. Further, we found a prominent reduction in Ser485 phosphorylation of AMP-dependent protein kinase (AMPK), and reductions in its target mTOR and downstream ribosomal protein S6 phosphorylation. We show that SAHA contributes most of the effect, as confirmed using another histone deacetylase inhibitor, trichostatin A. Overall, our results show that the combination of SAHA and doxorubicin can induce apoptosis in gastric adenocarcinoma in a synthetically lethal manner, and that fission yeast offers an efficient tool for identifying potent drug combinations against human cancer cells.

The Effect of the Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid and Paclitaxel Treatment on Full-Thickness Wound Healing in Mice.

Neoadjuvant chemotherapy prior to lumpectomy or mastectomy for breast cancer challenges wound healing. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been shown to work synergistically with paclitaxel in vitro and in preclinical studies. In addition, our laboratory has demonstrated that SAHA treatment decreases paclitaxel-associated stem cell toxicity, modulates inflammatory response, and promotes wound healing in injured fibroblast cells. Our goal was to determine if combined SAHA and paclitaxel treatment would improve wound healing in an in vivo full-thickness murine model, without altering antitumor effect. Thirty-two nude athymic mice received intraperitoneal injections of paclitaxel (20 mg/kg), SAHA (25 mg/kg), paclitaxel + SAHA (20 mg/kg + 25 mg/kg), or no treatment for 2 weeks prior to surgery. Under general anesthesia, 8-mm full-thickness dorsal wounds were created in all animals, and a silicone splint was attached to minimize wound contraction. The wounds were measured twice a week with a surgical caliper until healing was complete. To evaluate the in vivo effect of drug treatment, 16 athymic nude mice with MDA-MB-231 xenografts received the treatments described previously, following which tumor volumes were compared between groups. Average wound healing time was prolonged in mice treated with paclitaxel (20 ± 1.9 days), and combination SAHA + paclitaxel therapy improved average wound healing time (17.0 ± 1.8 days). In the xenograft model, the antitumor effect of SAHA and paclitaxel (average tumor volume 43.9 ± 34.1 mm) was greater than paclitaxel alone (105.8 ± 73.8 mm). The addition of SAHA to taxane chemotherapy improves the therapeutic effect on triple-negative breast cancer while decreasing the detrimental effect of paclitaxel on wound healing. This may have substantial implications on improving outcomes in breast reconstruction following chemotherapy.

Counteracting survival functions of EBNA3C in Epstein-Barr virus (EBV)-driven lymphoproliferative diseases by combination of SAHA and bortezomib.

Combination of suberoylanilide hydroxamic acid (SAHA) and bortezomib (SAHA/bortezomib) was shown to synergistically induce killing of lymphoblastoid cell lines (LCL) and Burkitt lymphoma (BL) of type III or Wp-restricted latency, both of which express EBNA3A, -3B and -3C proteins. We hypothesize that SAHA/bortezomib can counteract the survival functions conferred by the EBNA3 proteins. We tested the effect of SAHA/bortezomib on the survival of BL cell lines containing EBNA3A, -3B or -3C knockout EBV with or without the respective revertant EBNA3 genes. Isobologram analysis showed that SAHA/bortezomib induced significantly greater synergistic killing of EBNA3C-revertant cells when compared with EBNA3C-knockout cells. Such differential response was not observed in either EBNA3A or -3B revertant versus their knockout pairs. Interestingly, EBNA3C-knockout cells showed significant G2/M arrest whilst EBNA3C-revertant cells and LCLs escaped G2/M arrest induced by SAHA/bortezomib and became more susceptible to the induction of apoptosis. In parallel, SAHA/bortezomib induced stronger expression of p21WAF1 but weaker expression of p-cdc25c, an M-phase inducer phosphatase, in EBNA3C-expressing cells when compared with EBNA3C-knockout cells. SAHA/bortezomib also induced greater growth suppression of EBNA3C-expressing xenografts (EBNA3C-revertant and LCL) than that of EBNA3C-knockout xenografts in SCID mice. In conclusion, our data showed that SAHA/bortezomib could synergistically induce killing of BL and LCL through counteracting the survival functions of EBNA3C, providing a strong basis for clinical testing of this drug combination in patients with EBV-associated lymphoproliferative diseases.

A Combination of SAHA and Quinacrine Is Effective in Inducing Cancer Cell Death in Upper Gastrointestinal Cancers.

Purpose: We aimed to investigate the therapeutic efficacy of single agent and the combination of quinacrine and suberoylanilide hydroxamic acid (SAHA) in wt- and mut-p53 upper gastrointestinal cancer (UGC) cell models.Experimental Design: ATP-Glo, clonogenic cell survival, Annexin V, comet, DNA double-strand breaks (DSBs), qPCR, and Western blot analysis assays were utilized.Results: Using clonogenic cell survival, ATP-Glo cell viability, Annexin V, and sub-G0 population analysis, we demonstrated that a combination of quinacrine and SAHA significantly decreased colony formation and increased cancer cell death (range, 4-20 fold) in six UGC cell models, as compared with single-agent treatments, irrespective of the p53 status (P < 0.01). The combination of quinacrine and SAHA induced high levels of DSB DNA damage (>20-fold, P < 0.01). Western blot analysis showed activation of caspases-3, 9, and γ-H2AX in all cell models. Of note, although quinacrine treatment induced expression of wt-p53 protein, the combination of quinacrine and SAHA substantially decreased the levels of both wt-P53 and mut-P53. Furthermore, cell models that were resistant to cisplatin (CDDP) or gefitinib treatments were sensitive to this combination. Tumor xenograft data confirmed that a combination of quinacrine and SAHA is more effective than a single-agent treatment in abrogating tumor growth in vivo (P < 0.01).Conclusions: Our novel findings show that the combination of quinacrine and SAHA promotes DNA damage and is effective in inducing cancer cell death, irrespective of p53 status and resistance to CDDP or gefitinib in UGC models. Clin Cancer Res; 24(8); 1905-16. ©2018 AACR.

Enhanced anticancer efficacy of histone deacetyl inhibitor, suberoylanilide hydroxamic acid, in combination with a phosphodiesterase inhibitor, pentoxifylline, in human cancer cell lines and in-vivo tumor xenografts.

Vorinostat [suberoylanilide hydroxamic acid (SAHA)], a histone deacetylase inhibitor, shows limited clinical activity against solid tumors when used alone. The methyl xanthine drug, pentoxifylline (PENT), has been described to have antitumor properties. The aim of this study was to look for the enhanced anticancer activities of both agents when used in combination at doses lower than their respective efficacy dose when used alone. We investigated the antitumor potential of this novel combination in vitro and in vivo. The combination index was assessed for these two drugs to look for synergistic antiproliferative activity against a broad spectrum of human cancer cell lines. Consistent additive to synergistic interactions were observed in HCT116 cells when PENT was combined with SAHA at all drug tested concentrations. The combination of SAHA and PENT induces chromatin condensation and apoptosis downstream of the pan histone deacetylase inhibition and phosphodiesterase regulation, leading to subsequent cell cycle arrest at their lower tested concentrations. Further, the ability of this combination to inhibit angiogenesis, both in vitro and in vivo, was examined and a significant inhibition in tube formation in HUVEC cells and neovascularization of Matrigel plug was observed. A significant inhibition in tumor growth was observed in severe combined immunodeficient mice bearing HCT116 (colon) and PC3 (prostate) human xenografts treated with SAHA (30 mg/kg, intraperitoneal) in combination with PENT (60 mg/kg, intraperitoneal), with no loss in body weight and 100% survival. In conclusion, these findings indicate the enhanced anticancer activity of SAHA in combination with PENT both in vitro and in vivo.

Rescue of cell cycle progression in BRAFV600E inhibitor-resistant human melanoma by a chromatin modifier.

The BRAFV600E-specific inhibitor vemurafenib blocks mitogen-activated protein kinase pathway and induces cell cycle arrest at G0/G1 phase leading to apoptosis of melanomas. To gain an understanding of the dynamics of cell cycle regulation during vemurafenib therapy, we analyzed several vemurafenib-resistant human melanoma sublines derived from BRAFV600E harboring vemurafenib-sensitive parental lines. Vemurafenib provoked G0/G1 phase arrest in parental but not in vemurafenib-resistant sublines. We hypothesized that refractoriness of vemurafenib-resistant sublines to vemurafenib-mediated cell cycle inhibition can be partially rescued by the chromatin modifier suberoylanilide hydroxamic acid. Suberoylanilide hydroxamic acid promoted G2/M arrest at expense of S phase irrespective of vemurafenib sensitivity. In parental lines, combination of suberoylanilide hydroxamic acid and vemurafenib induced both G0/G1 arrest and apoptosis, whereas in vemurafenib-resistant sublines combination induced G0/G1 as well as G2/M arrest resulting in dramatic cytostasis. Vemurafenib-resistant sublines exhibited extracellular signal-regulated protein kinases 1 and 2 but not AKT and hyperphosphorylation. Gene expression profiling revealed mitogen-activated protein kinase hyperactivation and deregulations of cyclins and cyclin-dependent kinases in vemurafenib-resistant sublines, all of which were reversed by suberoylanilide hydroxamic acid; changes that may explain the cytostatic effects of suberoylanilide hydroxamic acid. These results suggest that unresponsiveness of vemurafenib-resistant sublines to the biological effects of vemurafenib may be amenable by suberoylanilide hydroxamic acid. These in vitro results, while require further investigation, may provide rational biological basis for combination therapy in the management of vemurafenib-resistant melanoma.