HDAC inhibitor HPTA initiates anti-tumor response by CXCL9/10-recruited CXCR3+CD4+T cells against PAHs carcinogenicity.

Polycyclic aromatic hydrocarbons (PAHs) exposure in food is closely associated with the occurrence and development of breast cancer, which may attribute to altered immunotoxicity and immune regulation. Currently, cancer immunotherapy aims to promote tumor-specific T cell responses, especially CD4+T helper cells (Th) for anti-tumor immunity. The histone deacetylase inhibitors (HDACis) are found to exert an anti-tumor effect by reshaping the tumor immune microenvironment, but the immune regulatory mechanism of HDACis in PAHs-induced breast tumor remains elusive. Here, using established breast cancer models induced by 7,12-dimethylbenz[a]anthracene (DMBA), a potent carcinogenic agent of PAH, the novel HDACi, 2-hexyl-4-pentylene acid (HPTA) exhibited anti-tumor effect by activating T lymphocytes immune function. HPTA recruited CXCR3+CD4+T cells into chemokines CXCL9/10-enriched tumor sites, and the increased secretion of CXCL9/10 was regulated by the NF-κB-mediated pathway. Furthermore, HPTA promoted Th1 differentiation and assisted cytotoxic CD8+T cells in the elimination of breast cancer cells. These findings support the proposition of HPTA as a potential therapeutic in the treatment of PAHs-induced carcinogenicity.

Screen identifies fasudil as a radioprotector on human fibroblasts.

Background: Radioprotectors safeguard biological system exposed to ionizing radiation (IR) by protecting normal cells from radiation damage during radiotherapy. Due to the toxicity and limited clinical utility of the present radioprotectors, it prompts us to identify novel radioprotectors that could alleviate IR-induced cytotoxicity of normal tissues. Aims and Methods: To identify new radioprotectors, we screened a chemical molecular library comprising 253 compounds in normal human fibroblasts (HFs) or 16HBE cells upon IR by CCK-8 assays and clonogenic survival assays. Fasudil was identified as a potential effective radioprotector. Results: The results indicated that Fasudil exerts radioprotective effects on HFs against IR-induced DNA double-strand breaks (DSBs) through the regulation of DSB repair. Fasudil increased homologous recombination (HR) repair by 45.24% and decreased non-homologous end-joining (NHEJ) by 63.88% compared with untreated cells, without affecting changes to cell cycle profile. We further found that fasudil significantly facilitated the expression and foci formation of HR core proteins such as Rad51 and BRCA1 upon IR, and decreased the expression of NHEJ-associated proteins such as DNA-PKcs at 24 h post-IR. Conclusion: Our study identified fasudil as a novel radioprotector that exert radioprotective effects on normal cells through regulation of DSB repair by promoting HR repair.

Valproic acid counteracts polycyclic aromatic hydrocarbons (PAHs)-induced tumorigenic effects by regulating the polarization of macrophages.

Polycyclic aromatic hydrocarbons (PAHs) are common persistent organic pollutants that are carcinogenic, teratogenic and mutagenic, causing a variety of harm to human health. In this study, we investigated the mechanism of how valproic acid (VPA) interferes with the carcinogenesis of PAHs protect normal tissues via the regulation of macrophages' function. Using the established model of transformed malignant breast cancer by 7,12-dimethylbenz[a]anthracene (DMBA), a representative PAH carcinogen, we discovered VPA induces the polarization of macrophages toward the M1 phenotype in the tumor tissues, facilitates the expression of pro-inflammatory cytokines such as IFN-γ, IL-12 and TNF-α, activates CD8+ T cells to secret Granzyme B thus to promote the apoptosis of tumor cells and suppresses the viability of vascular endothelial cells in tissue stroma of tumor. Surprisingly, VPA selectively induces macrophages to polarize towards the M2 phenotype in normal tissues and promotes the expression of anti-inflammatory cytokines such as IL-10 to enhance cell proliferation. Additionally, at the cellular level, VPA can directly regulate the polarization of macrophages to affect the growth of vascular endothelial cells by simulating the living conditions of tumor and normal cells. Collectively, VPA exerts an interventional effect on tumor growth and a protective effect on normal tissues by regulation of selective macrophages' polarization in their microenvironment.

Histone deacetylase inhibitor 2-hexyl-4-pentynoic acid enhances hydroxyurea therapeutic effect in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) treatment has only limited effect, and it causes a significant number of deaths. Histone deacetylase inhibitors (HDACis) are emerging as promising anti-tumor agents in many types of cancers. We thus hypothesized that 2-hexyl-4-pentynoic acid (HPTA), a novel HDACi, could sensitize TNBC to hydroxyurea (HU, a ribonucleotide reductase inhibitor). In the present study, we investigated the effect of HPTA, alone or in combination with HU on cell survival, DNA double-strand breaks (DSBs), key homologous recombination (HR) repair proteins and cell cycle progression in MDA-MB-468 and MDA-MB-231 human TNBC cell lines. HPTA and HU synergistically inhibited the survival of TNBC cell lines and resulted in the accumulation of DNA double-strand breaks (DSBs). HPTA can sensitize TNBC cells to HU by inhibiting replication protein A2 (RPA2) hyperphosphorylation-mediated HR repair, and lessen cell accumulation in S-phase by inhibiting ATR-CHK1 signaling pathway. Taken together, our data suggested that HPTA enhances HU therapeutic effect by blocking the HR repair and regulating cell cycle progression in TNBC.

Valproic Acid Regulates HR and Cell Cycle Through MUS81-pRPA2 Pathway in Response to Hydroxyurea.

Breast cancer is the primary problem threatening women's health. The combined application of valproic acid (VPA) and hydroxyurea (HU) has a synergistic effect on killing breast cancer cells, but the molecular mechanism remains elusive. Replication protein A2 phosphorylation (pRPA2), is essential for homologous recombination (HR) repair and cell cycle. Here we showed that in response to HU, the VPA significantly decreased the tumor cells survival, and promoted S-phase slippage, which was associated with the decrease of pCHK1 and WEE1/pCDK1-mediated checkpoint kinases phosphorylation pathway and inhibited pRPA2/Rad51-mediated HR repair pathway; the mutation of pRPA2 significantly diminished the above effect, indicating that VPA-caused HU sensitization was pRPA2 dependent. It was further found that VPA and HU combination treatment also resulted in the decrease of endonuclease MUS81. After MUS81 elimination, not only the level of pRPA2 was abolished in response to HU treatment, but also VPA-caused HU sensitization was significantly down-regulated through pRPA2-mediated checkpoint kinases phosphorylation and HR repair pathways. In addition, the VPA altered the tumor microenvironment and reduced tumor burden by recruiting macrophages to tumor sites; the Kaplan-Meier analysis showed that patients with high pRPA2 expression had significantly worse survival. Overall, our findings demonstrated that VPA influences HR repair and cell cycle through down-regulating MUS81-pRPA2 pathway in response to HU treatment.

Valproic Acid-Like Compounds Enhance and Prolong the Radiotherapy Effect on Breast Cancer by Activating and Maintaining Anti-Tumor Immune Function.

Inadequate sustained immune activation and tumor recurrence are major limitations of radiotherapy (RT), sustained and targeted activation of the tumor microenvironment can overcome this obstacle. Here, by two models of a primary rat breast cancer and cell co-culture, we demonstrated that valproic acid (VPA) and its derivative (HPTA) are effective immune activators for RT to inhibit tumor growth by inducing myeloid-derived macrophages and polarizing them toward the M1 phenotype, thus elevate the expression of cytokines such as IL-12, IL-6, IFN-γ and TNF-α during the early stage of the combination treatment. Meanwhile, activated CD8+ T cells increased, angiogenesis of tumors is inhibited, and the vasculature becomes sparse. Furthermore, it was suggested that VPA/HPTA can enhance the effects of RT via macrophage-mediated and macrophage-CD8+ T cell-mediated anti-tumor immunity. The combination of VPA/HPTA and RT treatment slowed the growth of tumors and prolong the anti-tumor effect by continuously maintaining the activated immune response. These are promising findings for the development of new effective, low-cost concurrent cancer therapy.

Valproic acid triggers radiation-induced abscopal effect by modulating the unirradiated tumor immune microenvironment in a rat model of breast cancer.

An abscopal effect occurs when localized radiotherapy causes the regression of tumors distant from the irradiated site. However, such a clinically detectable abscopal effect from radiotherapy alone is rare. This study investigated whether valproic acid ([VPA], a histone deacetylase inhibitor [HDACi]) treatment can stimulate radiation-induced abscopal effect. We used 7,12-dimethylbenz[a]anthracene, a typical environmental carcinogen, to establish a rat model with multiple breast tumors. Only one tumor received 8 Gy fractionated doses of X-rays (2 Gy daily fractions over four days) and 200 mg/kg VPA was administered intraperitoneally. We monitored the growth of both irradiated and unirradiated tumors after treatments. The unirradiated tumor was collected for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC) (CD8, Granzyme B, Cleaved Caspase-3, BrdU, Ki67, F4/80 and CD68), double immunofluorescence (F4/80 and CD86), Western blot (Cleaved Caspase-3) and qRT-PCR (CD86, CD163, IL-1ß, IL-6, IL-12, IL-23, IL-10, TGF-ß) analysis. We found ionizing radiation (IR) + VPA treatment inhibited both irradiated and unirradiated tumor growth as compared to IR alone. Such observe abscopal effect was mediated by the recruitment of activated CD8+ T cells into the unirradiated tumor sites, which released Granzyme B to cause tumor cell apoptosis. Furthermore, IR + VPA treatment led to macrophages infiltration into the unirradiated tumor sites and polarization to M1 phenotype, resulted in increased levels of pro-inflammatory cytokines such as IL-1ß and IL-12, and decreased levels of anti-inflammatory cytokines such as IL-10 and TGF-ß. Our data supports the proposition that VPA may be a potential therapeutic candidate to trigger radiation-induced abscopal effect by modulating the unirradiated tumor immune microenvironment.

The Valproate Mediates Radio-Bidirectional Regulation Through RFWD3-Dependent Ubiquitination on Rad51.

Ionizing radiation (IR) can induce DNA double-strand breaks (DSBs) in tumor cells during radiotherapy (RT), but the efficiency of RT is limited because of the toxicity to normal cells. Locating an adjuvant treatment to alleviate damage in normal cells while sensitizing tumor cells to IR has attracted much attention. Here, using the 7,12-dimethylbenz[α]anthracene (DMBA)-induced malignant transformed MCF10A cells, we found that valproate (VPA), a histone deacetylase inhibitor (HDACi), radiosensitized transformed cells while alleviated IR-induced damage in normal cells at a safe dose (0.5 mM). We further demonstrated the decrease of homologous recombination (HR)-associated Rad51 in the transformed cells was related to the increase of its ubiquitination regulated by E3 ligase RFWD3 for the radiosensitization, which was opposite to normal cells, indicating that RFWD3-dependent ubiquitination on Rad51 was involved in the VPA-mediated radio-bidirectional effect. Through DMBA-transformed breast cancer rat model, VPA at 200 mg/kg radiosensitized tumor tissue cells by increasing RFWD3 and inhibited Rad51, while radioprotected normal tissue cells by decreasing RFWD3 and enhanced Rad51. In addition, we found high-level Rad51 was associated with tumorigenesis and poor prognosis in breast cancer patients. Our findings uncovered RFWD3-dependent Rad51 ubiquitination was the novel mechanism of VPA-mediated radio-bidirectional effect, VPA is a potential adjuvant treatment for tumor RT.

The intervention of valproic acid on the tumorigenesis induced by an environmental carcinogen of PAHs.

This study investigated whether valproic acid (VPA, a histone deacetylase inhibitor) can interfere with the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs). A typical representative compound of PAHs, 7,12-Dimethylbenz[a]anthracene (DMBA), was used to induce rat breast cancer. The results showed that therapeutic concentration of VPA (50 and 100 mg/kg) delayed the occurrence of tumors, reduced tumor formation rate and attenuated tumors growth, and have a protective effect on normal tissues. The macrophage-mediated inflammatory response was found to be associated with the observed effect of VPA. In addition, we screened and validated a possible gene, Sema3c, which was involved in DMBA-induced breast cancer development and can be inhibited by VPA.

2-hexyl-4-pentynoic acid, a potential therapeutic for breast carcinoma by influencing RPA2 hyperphosphorylation-mediated DNA repair.

Breast carcinoma is one of the most common malignancies in women. Previous studies have reported that 500 µM valproic acid can sensitize breast tumor cells to the anti-neoplastic agent hydroxyurea. However, the dose requirements for valproic acid is highly variable due to the wide inter-individuals clinical characteristics. High therapeutic dose of valproic acid required to induce anti-tumor activity in solid tumor was associated with increased adverse effects. There are attempts to locate suitably high-efficient low-toxicity valproic acid derivatives. We demonstrated that lower dose of 2-hexyl-4-pentynoic acid (HPTA; 15 µM) has similar effects as 500 µM VPA in inhibiting breast cancer cell growth and sensitizing the tumor cells to hydroxyurea on MCF7 cells, EUFA423 cells, MCF7 cells with defective RPA2-p gene and primary culture cells derived from tissue-transformed breast tumor cells. We discovered HPTA resulted in more DNA double-strand breaks, the homologous recombination was inhibited through the interference of the hyperphosphorylation of replication protein A2 and recombinase Rad51. Our data postulate that HPTA may be a potential novel sensitizer to hydroxyurea in the treatment of breast carcinoma.

Valproic acid sensitizes breast cancer cells to hydroxyurea through inhibiting RPA2 hyperphosphorylation-mediated DNA repair pathway.

It was reported that valproic acid (VPA, a histone deacetylase inhibitor) can sensitize cancer cells to hydroxyurea (HU, a ribonucleotide reductase inhibitor) for chemotherapy, although the mechanism of VPA-induced HU sensitization is unclear. In this study, we systematically characterized VPA-induced HU sensitization of breast cancer cells. Multiple breast cancer cell models were employed to investigate whether the safe concentration of 0.5mM VPA and 2mM HU can result in DNA double-strand breaks (DSBs) and impact cell survival. Furthermore, the underlying mechanism was explored through cell biology assays, including clonogenic survival, homologous recombination (HR) activity, immunoblot and immunofluorescence. We found that VPA and HU cooperatively suppressed cancer cell survival. VPA resulted in the accumulation of more DNA double-strand breaks (DSBs) in response to HU-induced replication arrest and was able to block HU-stimulated homologous recombination (HR) through inhibiting the activity of two key HR repair proteins by hyperphosphorylation of replication protein A2 (RPA2-p) and recombinase Rad51. However, apoptosis was not detected under this condition. In addition, the results from the survival fraction in the cells expressing defective RPA2-p showed that VPA disrupted the HU-induced RPA2-p-Rad51-mediated HR pathway. Importantly, these findings were further supported by analyzing primary-culture cells from the tissue of chemical carcinogen (DMBA)-induced breast cancer in rats. Thus, our data demonstrated that VPA and HU synergistically suppressed tumor cells via disturbing RPA2-p-mediated DNA repair pathway, which provides a new way for combining chemotherapeutic drugs to sensitize breast cancer cells.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats.

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function.