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

Junxuan Peng; Zuchao Cai; Ruixue Zhao; Jiahao Chen; Guochao Liu; Chao Dong; David Lim; Zhihui Feng

doi:10.1093/toxres/tfaa069

. 2020 Aug 31;9(5):609–621. doi: 10.1093/toxres/tfaa069

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

Junxuan Peng ¹, Zuchao Cai ², Ruixue Zhao ³, Jiahao Chen ⁴, Guochao Liu ⁵, Chao Dong ⁶, David Lim ⁷, Zhihui Feng ^8,^✉

PMCID: PMC7640934 PMID: 33178421

Abstract

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.

Keywords: breast tumor, DMBA, valproic acid (VPA), macrophage, Sema3c

Graphical abstract

For DMBA-induced breast cancer in rats, VPA has the dual effects of interfering with tumor formation and protecting normal organs.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a kind of carcinogens that are commonly found in occupational and living environment. They are mainly derived from the incomplete combustion of organic substances. Research shows that PAHs not only has direct harm to the human body in special environment such as occupation, but also increases the risk of lung cancer, skin cancer and other malignant tumors in the long term [1]. It has been proved that the PAH 7,12-Dimethylbenz[a]anthracene (DMBA) is a potent carcinogen, and also the most commonly used carcinogen for inducing breast cancer and precancerous lesions in animals [2]. It has a strong selection for mammalian tissue with a specificity of 70 to 80%. Our studies and other research groups have shown that DMBA can successfully induce primary breast tumors in female Sprague Dawley (SD) rats [3, 4]. However, how to effectively intervene in the carcinogenic effects of PAHs has rarely been reported.

Valproic acid (VPA) is a short-chain fatty acid containing eight carbon atoms. It was synthesized by Burton in 1882 [5]. In 1963, Meunier’s group found that VPA has a strong antiepileptic effect and has been used since in clinical treatment. With the development of research on the role of VPA, it has been found that it has the effect of inhibiting histone deacetylase, which can inhibit the growth of a variety of tumor cells and even induce tumor cell death [6–8].

Some researches show that VPA can cause some tumors to be sensitive to radiotherapy or chemotherapy in vivo or in vitro, including thyroid cancer [9], liver cancer [10], glioma [11] and breast cancer [12]. In addition to its sensitization effect, VPA has recently been reported to have radioprotective effects on normal hippocampal neurons in vivo and in vitro [13–15]. So far, it was not clear whether VPA can prevent the PAHs-induced tumor development and protect the normal tissues.

The Semaphorins is a family of cell-surface soluble proteins, which can control the development of the central nervous system. The Sema3s subfamily is the only secreted protein in this family. A large number of studies have proved that the Sema3s protein subfamily not only regulates various functions of the nervous system, but also regulates the occurrence of tumors. Sema3c is an important member of the Sema3s protein subfamily [16–19]. Some studies have found that Sema3c is overexpressed in gastric cancer and liver cancer, and its expression level has a certain correlation with the stage and grade of tumors [20–22]. The overexpression of Sema3c in lung cancer cell lines significantly enhanced the metastatic ability of tumor cells [23]. Thus, it was indicated that Sema3c plays a role in promoting tumor development [20].

In this study, we used the DMBA-induced rat breast cancer model to explore whether VPA can interfere with the development of breast cancer and protect the normal tissues, and whether Sema3c is involved in the DMBA-induced breast cancer development and can be inhibited by VPA.

Materials and methods

The establishment of the animal model

Female SD rats were purchased from Peng Yue Laboratory Animal Co. Ltd., Jinan, China. The studies of animal tissue were performed in accordance with the requirements of the Shandong University Human and Animal Ethics Research Committee (project identification code 81472800, ethics approved 3 March 2014). All rats were housed in a specific pathogen-free environment (22 ± 1°C, 12-h light/dark cycle). Food and water were provided ad libitum. Rats were randomly divided into normal group, VPA (P4543, Sigma) 50 mg/kg group, VPA 100 mg/kg group, DMBA (D3254, Sigma) group, VPA 50 mg/kg intervention group and VPA 100 mg/kg intervention group. DMBA was dissolved in corn oil and adjusted to the concentration of 20 mg/ml. A single dose of 1 ml DMBA oil was administered to 50-day-old SD rats through intragastric gavage. At the second week after intragastric gavage, the rats were given VPA intraperitoneal at a dose of 50 mg/kg/day or 100 mg/kg/day for 105 consecutive days.

Tumor observation on rats

Two weeks after DMBA gavage, the mammary gland and adjacent parts in each rat of each group were observed, and the breast surrounding tissues were palpated. From the discovery of tumors, the tumor size, location and appearance of each group were recorded weekly and measured with Vernier Caliper. Tumor volume was calculated according to the clinical standard formula “Volume V (mm³) = Length (L) * Width (W)² * 0.5”.

BrdU incorporation and HE staining

5-Bromo-2′-deoxyuridine (BrdU) (B5002, Sigma) was injected intraperitoneally at a dose of 100 mg/kg 24 h before tissue harvest. Tumor tissues, normal breast and normal tissues were fixed overnight in 4% paraformaldehyde solution, embedded in paraffin and serially sectioned 5 μm thick for hematoxylin and eosin (HE) staining according to the manufacture’s procedures guideline.

Immunohistochemistry staining

The sections for immunohistochemistry (IHC) staining were dewaxed with xylene and hydrated with 100 to 75% ethanol, then boiled in a 10 mM sodium citrate solution at 92°C for 20 min for antigen retrieval. The sections were cooled to room temperature and then washed with tris-buffered saline (TBS). BrdU labeling requires DNA hydrolysis step: the sections were immersed in 1 M HCL for 1 h and then neutralized with 0.1 M sodium borate buffer (pH = 8.5) for 10 min. After washing with TBS, the sections were incubated in 3% hydrogen peroxide for 15 min to remove endogenous peroxidase and washed again with TBS. The treated sections were then incubated with 10% goat serum for 1 h to block nonspecific binding. Diluted primary antibodies were BrdU (1:50, Cat.347580, BD), F4/80 (1:300, 123102, Biolegend) and Sema3c (1:200, bs-6692R, Bioss). Secondary antibodies were biotinylated goat anti-mouse IgG (1:300, BA-9200, Vector) and biotinylated goat anti-rat IgG (1:300, BA-9400, Vector).

The relative number of BrdU and F4/80 positive cells stained by IHC was further quantified by Image pro plus software (Media Cybrenetics) and expressed by integral optical density (IOD).

RNA-seq

At the end of the experimental observation, the mammary gland tissues and tumor tissues of each group of rats were taken and rapidly frozen by liquid nitrogen for storage. The tissue samples were collected and sent to the KangChen Bio-tech company for RNA sequencing. There were three parallel samples for each group.

Real-time quantitative reverse transcriptase PCR

The genes with notable change were screened by RNA sequencing, using forward and reverse sequencing primers (Sangon Biotech). The specificity of the primers was verified by NCBI.

The RNA was extracted according to the RNA prep Pure Tissue Kit (Tiangen) protocol, and the isolated RNA was quantified by NanoDrop ND-2000 spectrophotometer (Nadro Drop Technologies, Wilmington, DE, USA). Complementary DNA (cDNA) synthesis was performed using the ReverAid First Strand cDNA Synthesis Kit (Thermo). Finally, specific primers and Maxima SYBR Green (Thermo) were used, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis was performed on Light Cycler® 480II (Roche Applied Science, Indianapolis, IN, USA) using 0.5 μL of each primer and 1 μL of cDNA.

The levels of the relative genes and the internal reference gene (GAPDH) expressed were measured, and the Ct values (threshold cycle number) of the target gene and the reference gene were calculated according to the Light Cycler® 480 Software release 1.5.0 SP4 software, use 2^{-ΔΔ Ct} method. Sample from the normal group mammary gland was used as control sample, and the expression of the target gene of each group was compared. ΔΔCt = experimental group ΔCt - control group ΔCt, ΔCt = (average Ct of the target gene of the control sample - average Ct of the control sample GAPDH) [24]. Primer sequences are listed in Table 1.

Table 1.

primers sequence of target genes

Oligo Name	Primer Sequence (5′-3′)
Sema3c-1	Forward: TGG GAA TTT CGT CCG CGT TA
	Reverse: ACG GTG TTC ACA TTG GGG TT
Sema3c-2	Forward: GCT CTA GGT TCT ACC CCA CT
	Reverse: TTT CAG ATT GAA CCC TCG GC
Slc6a14-1	Forward: GTG TGT TTG CTG GAT TCG CC
	Reverse: ACC ACC TGG GAG TTG AGC TA
Slc6a14-2	Forward: ATT GAA GTG CCC GAG CTT CT
	Reverse: GAC CAG TTA CCA CGC TCC TG
Pcp4	Forward: AGT TCA ACC ACC GTC CAT CC
	Reverse: GCA TTT CAG GTT AGC AGC GG
Adra1a	Forward: GCG GAG TCA GCA GTG CCA AG
	Reverse: GCC AGC AGA GGA CGA AGC AAC
Cdh17	Forward: ACC AAA CAC CCA AAG TTC CCT CAG
	Reverse: TGC TTC TTC AAG GAC CGT TTA CCC
Tdg	Forward: ACG CAA AGA GGA CGG CTG TTA AG
	Reverse: GCT CCG CAC TGT TGG CTG TTA G
Sox7	Forward: GCC ACC TAC CAC CCT CTC CAC
	Reverse: TCA AAC TCA TTG CGG TCC ATG TCC
Etv1	Forward: AGT CCA TAC CAG ACA GCA CCT ACC
	Reverse: CTT CCC TTG GCA TTG TTG GCA AAG
Ncmap	Forward: TCG TGG TGG TTG TCG TCA TCA TTG
	Reverse: CAG GTG GCT TTG GGC TCT TGG
Adhfe1	Forward: ATT CGC ACC GCC AAG ATC CAA G
	Reverse: GGC AGC GAG ACC GTC ATC AAC
Atp13a4	Forward: ACT TCG TTG TGT CCC TTG CTG TG
	Reverse: CTT GAA TCA GTC GCC AGG TTC CTC
Pcp4l1	Forward: AGG CCG AGG AGG AGG AAG AA
	Reverse: CGG AAC TTG CCC TGG ATT GC
Ech1	Forward: GGG CTG TTG TGG TCT CTG GT
	Reverse: GGG GCT GCA GGA TGT CTG AT
Mmp13	Forward: ATA CGA GCA TCC ATC CCG AGA CC
	Reverse: AAC CGC AGC ACT GAG CCT TTT C
R3hdml	Forward: TTA AGG ATG CCC AAC ACC ACA CTG
	Reverse: AAT CCA GCA AGG CGT TCA TAT CCC
Pcdh1	Forward: GAG ACA GGC AGC GAC AGC AAG
	Reverse: GTC CAC CGC CAC GAT CTC AAT G
Dcbld2	Forward: CAA GCA CTC CAA CCA CGA AGT AGG
	Reverse: GAC CAG CAC AGG AAC CAG AAC G
Jakmip1	Forward: TGC TGC GTG GGT AGA GGA GAA G
	Reverse: CGT TCT GGT CCT TGG CGT CTT G
GAPDH	Forward: CTC ATG ACC ACA GTC CAT GC
	Reverse: TTC AGC TCT GGG ATG ACC TT

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Western blot analysis

Breast tissues and tumors were homogenized in lysis buffer with protease inhibitors and clarified by centrifugation. The protocols were described in our previous publication [25]. The primary antibodies include Sema3c (1:500, bs-6692R, Bioss) and GAPDH (1:2000, TA-08, ZSGB-BIO). The secondary antibodies were goat anti-mouse lgG (H + L) (1:5000, 31430, Thermo Fisher) and goat anti-rabbit lgG (H + L) (1:5000, 31460, Thermo Fisher). The amounts of protein relative to the loading control were quantified by ImageJ software.

Statistical analysis

The results are expressed as means ± standard deviation for the groups. Data were analyzed by independent sample t-test. P < 0.05 or P < 0.01 indicated a statistically significant difference.

Results

VPA can reduce DMBA-induced tumor formation in rats

As shown in Fig. 1A, the neoplasms were found in the breast of the rats at around 40 days after the application of DMBA by intragastric gavage [26]. Through HE staining (Fig. 1B), it was shown that there were a few mammary ducts and lots of loose connective tissue in normal breast tissue, while in the tissue of DMBA group, there was a lot of pathological dysplasia and interstitial fibrosis, which was consistent with other reports [3,4,27], indicating the breast cancer model on rats was successfully established.

VPA can reduce the rate of tumor formation and growth in rats. (A) The establishment of DMBA-induced breast cancer in rat. The pictures presented the normal breast (1) and DMBA-induced breast cancer (2 and 3). (B) HE staining for the morphology of normal tissue (1) and DMBA induced breast cancer (2 and 3). (C) The schedule of DMBA exposure and VPA administration. 50mg/kg/day and 100mg/kg/day VPA (*i.p.*) injection were performed for 105 consecutive days after DMBA exposure. (D) The graph about the earliest time of tumor formation in each group. (E) The graph about the tumor formation rate of each group. (F) The graph about the percentage of tumor formation in the rats with different patterns divided by the number of tumors. (G) Representative images show tumor morphology of each group (left), the graph about standardized tumor volume in each group (right). Notes: Each data point in the graph was from three independent experiments (mean ± SD); P-Values were calculated by t-test (^*P < 0.05, ^**P < 0.01).

VPA intervention was performed at 2 weeks after DMBA application, the intervention schedule of VPA was shown in Fig. 1C, two doses of VPA were used: 50 and 100 mg/kg/day by intraperitoneal injection. At around 40 days after gavage, tumor appeared only in DMBA group, while tumors appeared at around 57th day or 63rd day in the 50 mg/kg- or 100 mg/kg-VPA intervention group (Fig. 1D), which was a delay of 17 days or 23 days in the 50 mg/kg- or 100 mg/kg-VPA intervention group as compared with the DMBA group (P < 0.05). However, there was no tumor formation in the control group and VPA-only negative control group over the course of the observation. The results suggested that VPA can delay the occurrence of breast tumors in a dose-dependent manner.

Comparing the formation rate of tumors across the groups at the end of the observation period (Fig. 1E), we found that the tumor formation rate was 70.56% in the DMBA group, 55.56% in the 50 mg/kg and 44.44% in the 100 mg/kg-VPA intervention group. The rate of tumor formation decreased by 15 and 26.12% in both VPA intervention groups, 50 and 100 mg/kg, respectively (P < 0.01), indicating that VPA was also able to inhibit the formation of tumors in a dose-dependent manner.

We further observed that the tumor formation rate in rats with more than three tumors in the DMBA group was 42.06%, while the rate was 0% in the 50 mg/kg and 16.67% in the 100 mg/kg VPA groups (Fig. 1F, P < 0.05). The data demonstrated that VPA can reduce multiple tumor formation.

We next investigated whether VPA can influence the tumor growth, hence the tumor volume was measured and compared across the treatment groups. As shown in Fig. 1G, the tumor size in both VPA intervention groups (50 and 100 mg/kg) was significantly smaller than that of the DMBA group (Fig. 1G left), which was consistent with the results of Fig. 1E. Moreover, the tumor growth rate in both VPA intervention groups was also significantly slower than that of DMBA group (Fig. 1G right, P < 0.01). However, there was no significant difference between the two VPA intervention groups (P > 0.05).

VPA can inhibit the proliferation of DMBA-induced tumor cells in rats

Firstly, the tissue morphological structure in each group was observed by HE staining (Fig. 2A). It was shown that there was no significant difference between the VPA-only and the normal group, a small amount of mammary duct and a large amount of loose connective tissue were seen in the groups. In DMBA group, pathological abnormal hyperplasia, a great amount of malignant cells and interstitial fibrosis were exhibited, while vacuoles necrosis appeared in the tumor tissue after VPA intervention, indicating that VPA can result in the necrosis of tumor tissue.

Secondly, we further investigated whether VPA can affect the ability of cell proliferation in tumor using the proliferative marker bromodeoxyuridine (BrdU) [28]. As shown in Fig. 2B, some proliferative cells in breast tissue appeared in the control and VPA-only groups, while a large number of proliferating cells in tumor tissue appeared in the DMBA group. The VPA intervention resulted in a dramatic decrease in proliferating cells in tumor tissues, regardless of both VPA intervention groups (50 and 100 mg/kg), suggesting VPA was able to suppress tumor cell proliferation.

Recently, it was reported that TMP195, a HDACi [29] could stimulate and recruit macrophages to tumor sites to suppressing tumor proliferation. Therefore, we further investigated whether VPA is also able to induce macrophage activity in our model. The specific macrophage marker F4/80 [30] was employed to observe the number and distribution in the tissues by immunohistochemical staining (Fig. 2C). Compared with the DMBA group, there were more macrophages in both VPA intervention groups, especially in the 100 mg/kg-VPA intervention group, and they were mainly located in the vacuole regions formed by tumor necrosis, indicating that VPA may stimulate and recruit macrophages to tumor sites for the killing of tumor cells.

VPA had a protective effect on normal tissues in DMBA-exposed rats

To test whether VPA intervention has any toxic effects on non-tumor tissues on rats, the following experiments were further conducted. At first, the rats’ weight in each of the groups was measured (Fig. 3A). We found that the rat weight in DMBA group slowed down significantly in the middle and later stage of the observation, while that the rat weight in both VPA intervention groups recovered, similar to that in normal rats, potentially suggesting that VPA may have no toxic effect to normal tissues, in contrast to the effect of VPA to tumor, supporting the proposition that VPA has a protective effect against normal tissues in rats.

We next measured the organ index of each group, such as liver, lung, spleen and brain, and found that there was no significant difference between the groups (Fig. 3B). We also found no significant change in the tissue morphology in spleen, small intestine, lung, liver and brain in each group by HE staining (Fig. 3C).

(C) The pictures presented the morphological structure in spleen, small intestine, lung, liver and brain of each group by HE staining.

As shown in Fig. 3D, despite no statistically significant difference in the organ index and morphology, we used BrdU labeling to detect the cellular proliferation in spleen and small intestine. We found no significant difference between the VPA-only group and the normal group (P > 0.05), the proliferation significantly decreased in the DMBA group as compared to control group (P < 0.01), and VPA was able to recover the proliferation ability in these tissues (P < 0.01). Our data suggest that VPA may have a protective effect against DMBA-induced normal tissue injury.

(D) The pictures presented the proliferated cells of spleen and small intestine stained by IHC with specific antibody BrdU (left), the relative number of BrdU positive cells was further quantified by Image pro plus software and expressed by IOD density (right); (E) The pictures presented the macrophages of spleen and small intestine stained by IHC with specific antibody F4/80 (left), the relative number of F4/80 positive cells was further quantified by Image pro plus software and expressed by IOD density (right). Notes: Each data point in the graph was from three independent experiments (mean ± SD); P-Values were calculated by t-test (^*P < 0.05, ^**P < 0.01).

By IHC staining with F4/80 antibody (Fig. 3E), macrophages infiltration in the spleen and small intestine was observed in the DMBA group (P < 0.01), indicating the decease of cell proliferation ability may be associated with the macrophage-induced inflammatory damage. The infiltrated macrophages in the spleen and small intestine in both VPA intervention groups were significantly reduced (P < 0.01), which further indicated that VPA had a protective effect on spleen and small intestine for DMBA-exposed rats, and could be achieved by reducing macrophage-mediated inflammation injury. In addition, in other organs (such as lung and brain, data not shown), there was no difference in the results from BrdU and F4/80 staining experiments in each group.

The analysis of the potential genes associated with DMBA-induced tumorigenesis

To find the key genes in the development of DMBA-induced breast cancer, we next performed RNA-Seq analysis on the tissues in each group. It was supposed that the key genes would have Type I or II properties. Type I: expression of some genes upregulated in DMBA as compared to the control group, and down-regulated in the VPA combination group. Type II: expression of some genes down-regulated in DMBA as compared to the control group, and upregulated in the VPA combination group. Through the RNA-Seq analysis, the Type I and II genes identified were tabulated in Table 2.

Table 2.

the genes potentially involved in DMBA-induced tumorigenesis through RNA-seq analysis

Up DMBA vs Normal Down DMBA+VPA vs DMBA (Type I)		Down DMBA vs Normal Up DMBA+VPA vs DMBA (Type II)
Mmp13	Sema3c	Pcp4
R3hdml	Cdh17	Adhfe1
Slc6a14	Tdg	Atp13a4
Pcdh1	Sox7	Pcp4l1
Dcbld2	Etv1	Ech1
Jakmip1	Ncmap
Adra1a

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VPA potentially inhibit the formation of DMBA-induced tumors by interfering with the expression of tumor-associated genes Sema3c

According to the literature review the following three genes: Sema3c, Slc6a14 and Pcp4 are closely related to the formation of breast cancer. We next used qRT-PCR analysis to confirm the RNA-seq results. The data showed that compared with normal mammary tissues, the expression of Sema3c in DMBA-induced group was increased (Fig. 4A, P < 0.01), consistent with Cole Healy Z [31] and TCGA mammary database (Fig. 4B). In the VPA combination therapy group, particularly in the 100 mg/kg-VPA intervention group, the expression of Sema3c was significantly reduced (Fig. 4A, P < 0.05), the data about the other two genes (Slc6a14 and Pcp4) showed no statistically significant relationship with breast cancer (Fig. 4C and D, P > 0.05). We next validated the other 15 genes in Table 2 by qRT-PCR and found that except Atp13a4, Adhfe1 and Tdg, the other results were consistent with the results of sequencing (Fig. S1). To address the important role of Sema3c in DMBA-induced breast tumors in rat, we also explore the protein level of Sema3c. As shown in Fig. 4E, through IHC staining, there were almost no Sema3c-positive cells in the control and VPA-only groups, while a large number of Sema3c-positive cells in tumor tissue appeared in the DMBA group. The VPA intervention resulted in a dramatic decrease in tumor tissues, regardless of VPA doses. Furthermore, using western blot (Fig. 4F), we found that the protein level of Sema3c in the DMBA group is inhibited by VPA treatment.

VPA may inhibit the formation of breast tumors by interfering with the expression of Sema3c (A) The graph about relative mRNA expression of Sema3c tested by qRT-PCR; (B) The graph about Sema3c expression in Oncomine Breast Statistics; (C) The graph about relative mRNA expression of Slc6a14 tested by qRT-PCR; (D) The graph about relative mRNA expression of Pcp4 tested by qRT-PCR. (E) The pictures presented the Sema3c positive cells in breast and tumor tissues stained by IHC with specific antibody Sema3c (left), the relative number of Sema3c positive cells was further quantified by Image pro plus software and expressed by IOD density (right); (F) The level of Sema3c protein in breast and tumor tissues was detected by immunoblotting (left), and the density was further analyzed by the Image J (right). Notes: Each data point in the graph was from three independent experiments (mean ± SD); P-Values were calculated by t-test (^*P < 0.05, ^**P < 0.01).

Our above results suggest that Sema3c may play a key role in DMBA-induced tumorigenesis and VPA can interrupt tumorigenesis through the inhibition of Sema3c expression.

Discussion

In this study, we have found that VPA can significantly reduce the incidence of DMBA-induced breast tumors in rats. Although VPA could not completely inhibit tumor growth, it was observed that VPA can slow down the growth rate of tumors and reduce the volume and number of tumors. Therefore, our study affirmed that VPA can prevent DMBA-induced tumor growth and delaying its growth.

Currently, the following four aspects about the carcinogenic mechanisms of PAHs are reported: (1) PAHs: it can affect the activity of PAHs by related enzymes such as CYP450, GST, epoxide hydrolase and the formation of PAHs-DNA adducts carcinogenic [32–34]; (2) it is also possible that PAHs can regulate cell proliferation, differentiation, and apoptosis by acting on an aryl hydrocarbon receptor (AhR) to cause malignant tumor formation [35]; (3) some studies have shown that PAHs enter the human body and can induce inflammation in a variety of tissues and organs, resulting in a cancerous microenvironment [36], which in turn leads to tumorigenesis; (4) it is considered that PAHs may cause cancer through epigenetic mechanisms, resulting in reduced genome methylation, increased methylation of tumor suppressor genes, and decreased methylation of proto-oncogenes [37].

Through RNA-seq analysis, we firstly screened and found Semaphorin 3C (Sema3c) to be also closely related to the development of DMBA-induced breast cancer. Sema3c is a member of the Semaphorin family and plays an important role in many physiological processes, including axonal growth, immune response, cell adhesion, migration and bone remodeling [38]. Malik’s group had previously mentioned that higher Sema3c expression was associated with tumor differentiation and increases adhesion and invasion of breast cancer cells [39], suggesting that this gene may be a potential target for PAHs-induced breast cancer treatment. Herman’s group had also shown that Sema3c can promote tumorigenesis by promoting endothelial cell migration, cancer metastasis and angiogenesis, and then promote tumorigenesis [39, 40]. Our results demonstrated that Sema3c can be upregulated in DMBA-induced breast cancer, and VPA can inhibit its expression during tumor development. At the same time, the qRT-PCR showed that treatment with low dose of VPA (50 mg/kg) can upregulate the expression of Sema3c in rat mammary tissues at the transcription level, and did not affect the expression at the protein level. The precise involvement and role Sema3c played in the oncogenesis induced by DMBA, and how to regulate it will need further verification. In addition, the two genes, Slc6a14 and Pcp4, obtained by RNA-seq analysis, have not been proved to be related to DMBA-induced breast cancer in the qRT-PCR experiment. Considering the rigor of the experiment, IHC staining can be carried out to confirm the expression of these two proteins, which needs to be further implemented.

Besides detecting the three genes mentioned above, we further validated 15 other genes by qRT-PCR, and the mRNA expression levels of 12 genes were consistent with the results of RNA-seq. Some scholars reported that they may be involved in the invasion, transformation, proliferation and migration of the tumors, such as gastric cancer [41], pancreatic cancer [42], colorectal cancer [43], etc. Therefore, these are also worthy of further exploration.

On the intervention study of DMBA carcinogenesis of PAHs, Mohamed’s group [44] found that antioxidants can prevent breast cancer induced by DMBA. In our study, VPA induced the infiltration of macrophages in the tumor tissue, potentially suggest that VPA-stimulated inflammatory response is involved in the intervention of breast cancer formation, this phenomenon was previously observed with another HDACi (TMP195) [29]. Meanwhile, our study also found VPA can protect DMBA-induced normal organ’s damage by reducing macrophage-mediated inflammatory response, such as for spleen and small intestine. The dual effects of VPA to regulate tumor tissue and normal tissues still need to be further illustrated. In summary, our study provides theoretical support and evidence for the intervention of PAHs-involved tumorigenesis after occupational exposure.

Conclusion

Collectively, the findings of the present study reveal that for DMBA-induced breast cancer in rats, VPA can play an effective role in interfering with tumor formation by inhibiting Sema3c. In addition, we also found the protective effect of VPA on normal tissues and speculated that macrophages play a very important role in this dual effect. These observations provide foundational knowledge that could be utilized for in the development of novel preventive strategies to allow for the protection against the tumors that occurs as a result of exposure of DMBA or other PAHs.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Supplementary Material

FigureS1_tfaa069

Click here for additional data file.^{(1.2MB, png)}

Acknowledgments

This research was supported by grants from National Natural Science Foundation of China (No. 81472800), and Department of Science and Technology of Shandong Provence (2019GSF108083).

Contributor Information

Junxuan Peng, Department of Occupational Health and Occupational Medicine, School of Public Health, Cheeloo College of Medicine, Shandong University, 44 Wenhua Xi Road, Shandong, Jinan, 250012, China.

Zuchao Cai, Department of Occupational Health and Occupational Medicine, School of Public Health, Cheeloo College of Medicine, Shandong University, 44 Wenhua Xi Road, Shandong, Jinan, 250012, China.

Ruixue Zhao, Department of Occupational Health and Occupational Medicine, School of Public Health, Cheeloo College of Medicine, Shandong University, 44 Wenhua Xi Road, Shandong, Jinan, 250012, China.

Jiahao Chen, Department of Occupational Health and Occupational Medicine, School of Public Health, Cheeloo College of Medicine, Shandong University, 44 Wenhua Xi Road, Shandong, Jinan, 250012, China.

Guochao Liu, Department of Occupational Health and Occupational Medicine, School of Public Health, Cheeloo College of Medicine, Shandong University, 44 Wenhua Xi Road, Shandong, Jinan, 250012, China.

Chao Dong, Department of Occupational Health and Occupational Medicine, School of Public Health, Cheeloo College of Medicine, Shandong University, 44 Wenhua Xi Road, Shandong, Jinan, 250012, China.

David Lim, School of Science and Health, Western Sydney University, Narellan Road, Campbelltown NSW 2560, Australia.

Zhihui Feng, Department of Occupational Health and Occupational Medicine, School of Public Health, Cheeloo College of Medicine, Shandong University, 44 Wenhua Xi Road, Shandong, Jinan, 250012, China.

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6. Nolan L, Johnson PW, Ganesan A et al. Will histone deacetylase inhibitors require combination with other agents to fulfil their therapeutic potential? Br J Cancer 2008;99:689–94. doi: 10.1038/sj.bjc.6604557. [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Wagner JM, Hackanson B, Lubbert M et al. Histone deacetylase (HDAC) inhibitors in recent clinical trials for cancer therapy. Clin Epigenetics 2010;1:117–36. doi: 10.1007/s13148-010-0012-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Sharma NL, Groselj B, Hamdy FC et al. The emerging role of histone deacetylase (HDAC) inhibitors in urological cancers. BJU Int 2013;111:537–42. doi: 10.1111/j.1464-410X.2012.11647.x. [DOI] [PubMed] [Google Scholar]
9. Perona M, Thomasz L, Rossich L et al. Radiosensitivity enhancement of human thyroid carcinoma cells by the inhibitors of histone deacetylase sodium butyrate and valproic acid. Mol Cell Endocrinol 2018;478:141–50. doi: 10.1016/j.mce.2018.08.007. [DOI] [PubMed] [Google Scholar]
10. Yu JI, Choi C, Shin SW et al. Valproic acid sensitizes hepatocellular carcinoma cells to proton therapy by suppressing NRF2 activation. Sci Rep 2017;7:14986. doi: 10.1038/s41598-017-15165-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Chang YL, Huang LC, Chen YC et al. The synergistic effects of valproic acid and fluvastatin on apoptosis induction in glioblastoma multiforme cell lines. Int J Biochem Cell Biol 2017;92:155–63. doi: 10.1016/j.biocel.2017.10.003. [DOI] [PubMed] [Google Scholar]
12. Tian Y, Liu G, Wang H et al. Valproic acid sensitizes breast cancer cells to hydroxyurea through inhibiting RPA2 hyperphosphorylation-mediated DNA repair pathway. DNA Repair (Amst) 2017;58:1–12. doi: 10.1016/j.dnarep.2017.08.002. [DOI] [PubMed] [Google Scholar]
13. Thotala D, Karvas RM, Engelbach JA et al. Valproic acid enhances the efficacy of radiation therapy by protecting normal hippocampal neurons and sensitizing malignant glioblastoma cells. Oncotarget 2015;6:35004–22. doi: 10.18632/oncotarget.5253. [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Zhang H, Zhang W, Zhou Y et al. Dual functional mesoporous silicon nanoparticles enhance the radiosensitivity of VPA in glioblastoma. Transl Oncol 2017;10:229–40. doi: 10.1016/j.tranon.2016.12.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Liao G, Li R, Chen X et al. Sodium valproate prevents radiation-induced injury in hippocampal neurons via activation of the Nrf2/HO-1 pathway. Neuroscience 2016;331:40–51. doi: 10.1016/j.neuroscience.2016.06.019. [DOI] [PubMed] [Google Scholar]
16. Scott GA, McClelland LA, Fricke AF. Semaphorin 7a promotes spreading and dendricity in human melanocytes through beta1-integrins. J Invest Dermatol 2008;128:151–61. doi: 10.1038/sj.jid.5700974. [DOI] [PubMed] [Google Scholar]
17. Kumanogoh A, Kikutani H. Roles of the semaphorin family in immune regulation. Adv Immunol 2003;81:173–98. doi: 10.1016/s0065-2776(03)81005-2. [DOI] [PubMed] [Google Scholar]
18. Roth JM, Kohler D, Schneider M et al. Semaphorin 7A aggravates pulmonary inflammation during lung injury. PLoS One 2016;11:e0146930. doi: 10.1371/journal.pone.0146930. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Mizutani N, Nabe T, Yoshino S. Semaphorin 7A plays a critical role in IgE-mediated airway inflammation in mice. Eur J Pharmacol 2015;764:149–56. doi: 10.1016/j.ejphar.2015.07.004. [DOI] [PubMed] [Google Scholar]
20. Bussolino F, Giraudo E, Serini G. Class 3 semaphorin in angiogenesis and lymphangiogenesis. Chem Immunol Allergy 2014;99:71 –88. doi: 10.1159/000353315. [DOI] [PubMed] [Google Scholar]
21. Nakamura F, Kalb RG, Strittmatter SM. Molecular basis of semaphorin-mediated axon guidance. J Neurobiol 2000;44:219–29. doi: . [DOI] [PubMed] [Google Scholar]
22. Potiron V, Roche J. Class 3 semaphorin signaling: the end of a dogma. Sci STKE 2005;2005:pe24. doi: 10.1126/stke.2852005pe24. [DOI] [PubMed] [Google Scholar]
23. Yoshida Y. Semaphorin signaling in vertebrate neural circuit assembly. Front Mol Neurosci 2012;5:71. doi: 10.3389/fnmol.2012.00071. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Talhada D, Goncalves I, Reis Santos C et al. Transthyretin expression in the postischemic brain. PLoS One 2019;14:e0221555. doi: 10.1371/journal.pone.0221555. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Feng Z, Scott SP, Bussen W et al. Rad52 inactivation is synthetically lethal with BRCA2 deficiency. Proc Natl Acad Sci U S A 2011;108:686–91. doi: 10.1073/pnas.1010959107. [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Liu G, Wang H, Zhang F et al. The effect of VPA on increasing radiosensitivity in osteosarcoma cells and primary-culture cells from chemical carcinogen-induced breast cancer in rats. Int J Mol Sci 2017;18. doi: 10.3390/ijms18051027. [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Pugalendhi P, Manoharan S, Suresh K et al. Genistein and daidzein, in combination, protect cellular integrity during 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary carcinogenesis in Sprague-Dawley rats. Afr J Tradit Complement Altern Med 2011;8:91–7. doi: 10.4314/ajtcam.v8i2.63196. [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Beresford MJ, Wilson GD, Makris A. Measuring proliferation in breast cancer: practicalities and applications. Breast Cancer Res 2006;8:216. doi: 10.1186/bcr1618. [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Guerriero JL, Sotayo A, Ponichtera HE et al. Class IIa HDAC inhibition reduces breast tumours and metastases through anti-tumour macrophages. Nature 2017;543:428–32. doi: 10.1038/nature21409. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Dos Anjos Cassado A. F4/80 as a major macrophage marker: the case of the peritoneum and spleen. Results Probl Cell Differ 2017;62:161–79. doi: 10.1007/978-3-319-54090-0_7. [DOI] [PubMed] [Google Scholar]
31. Cole-Healy Z, Vergani P, Hunter K et al. The relationship between semaphorin 3C and microvessel density in the progression of breast and oral neoplasia. Exp Mol Pathol 2015;99:19–24. doi: 10.1016/j.yexmp.2015.03.041. [DOI] [PubMed] [Google Scholar]
32. Xue W, Warshawsky D. Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: a review. Toxicol Appl Pharmacol 2005;206:73–93. doi: 10.1016/j.taap.2004.11.006. [DOI] [PubMed] [Google Scholar]
33. Farrell G. Effects of disease on expression and regulation of CYPs. Mol Aspects Med 1999;20:55–70137. [PubMed] [Google Scholar]
34. Gallagher EP, Wienkers LC, Stapleton PL et al. Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin B1. Cancer Res 1994;54:101–8. [PubMed] [Google Scholar]
35. Gasiewicz TA, Henry EC, Collins LL. Expression and activity of aryl hydrocarbon receptors in development and cancer. Crit Rev Eukaryot Gene Expr 2008;18:279–321. doi: 10.1615/critreveukargeneexpr.v18.i4.10. [DOI] [PubMed] [Google Scholar]
36. Fitzpatrick FA. Inflammation, carcinogenesis and cancer. Int Immunopharmacol 2001;1:1651–67. doi: 10.1016/s1567-5769(01)00102-3. [DOI] [PubMed] [Google Scholar]
37. Wilson AS, Power BE, Molloy PL. DNA hypomethylation and human diseases. Biochim Biophys Acta 2007;1775:138–62. doi: 10.1016/j.bbcan.2006.08.007. [DOI] [PubMed] [Google Scholar]
38. Rizzolio S, Tamagnone L. Semaphorin signals on the road to cancer invasion and metastasis. Cell Adh Migr 2007;1:62–8. doi: 10.4161/cam.1.2.4570. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Malik MF, Satherley LK, Davies EL et al. Expression of Semaphorin 3C in breast cancer and its impact on adhesion and invasion of breast cancer cells. Anticancer Res 2016;36:1281–6. [PubMed] [Google Scholar]
40. Herman JG, Meadows GG. Increased class 3 semaphorin expression modulates the invasive and adhesive properties of prostate cancer cells. Int J Oncol 2007;30:1231–8. [PubMed] [Google Scholar]
41. Chen Y, Low TY, Choong LY et al. Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells. Proteomics 2007;7:2384–97. doi: 10.1002/pmic.200600968. [DOI] [PubMed] [Google Scholar]
42. Heeg S, Das KK, Reichert M et al. ETS-transcription factor ETV1 regulates stromal expansion and metastasis in pancreatic cancer. Gastroenterology 2016;151:540–553 e514. doi: 10.1053/j.gastro.2016.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Tian X, Han Z, Zhu Q et al. Silencing of cadherin-17 enhances apoptosis and inhibits autophagy in colorectal cancer cells. Biomed Pharmacother 2018;108:331–7. doi: 10.1016/j.biopha.2018.09.020. [DOI] [PubMed] [Google Scholar]
44. Zeweil MM, Sadek KM, Taha NM et al. Graviola attenuates DMBA-induced breast cancer possibly through augmenting apoptosis and antioxidant pathway and downregulating estrogen receptors. Environ Sci Pollut Res Int 2019;26:15209–17. doi: 10.1007/s11356-019-04920-w. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

FigureS1_tfaa069

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[ref1] 1. Zhang Y, Tao S, Shen H et al. Inhalation exposure to ambient polycyclic aromatic hydrocarbons and lung cancer risk of Chinese population. Proc Natl Acad Sci U S A 2009;106:21063–7. doi: 10.1073/pnas.0905756106. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[ref8] 8. Sharma NL, Groselj B, Hamdy FC et al. The emerging role of histone deacetylase (HDAC) inhibitors in urological cancers. BJU Int 2013;111:537–42. doi: 10.1111/j.1464-410X.2012.11647.x. [DOI] [PubMed] [Google Scholar]

[ref9] 9. Perona M, Thomasz L, Rossich L et al. Radiosensitivity enhancement of human thyroid carcinoma cells by the inhibitors of histone deacetylase sodium butyrate and valproic acid. Mol Cell Endocrinol 2018;478:141–50. doi: 10.1016/j.mce.2018.08.007. [DOI] [PubMed] [Google Scholar]

[ref10] 10. Yu JI, Choi C, Shin SW et al. Valproic acid sensitizes hepatocellular carcinoma cells to proton therapy by suppressing NRF2 activation. Sci Rep 2017;7:14986. doi: 10.1038/s41598-017-15165-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref11] 11. Chang YL, Huang LC, Chen YC et al. The synergistic effects of valproic acid and fluvastatin on apoptosis induction in glioblastoma multiforme cell lines. Int J Biochem Cell Biol 2017;92:155–63. doi: 10.1016/j.biocel.2017.10.003. [DOI] [PubMed] [Google Scholar]

[ref12] 12. Tian Y, Liu G, Wang H et al. Valproic acid sensitizes breast cancer cells to hydroxyurea through inhibiting RPA2 hyperphosphorylation-mediated DNA repair pathway. DNA Repair (Amst) 2017;58:1–12. doi: 10.1016/j.dnarep.2017.08.002. [DOI] [PubMed] [Google Scholar]

[ref13] 13. Thotala D, Karvas RM, Engelbach JA et al. Valproic acid enhances the efficacy of radiation therapy by protecting normal hippocampal neurons and sensitizing malignant glioblastoma cells. Oncotarget 2015;6:35004–22. doi: 10.18632/oncotarget.5253. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref14] 14. Zhang H, Zhang W, Zhou Y et al. Dual functional mesoporous silicon nanoparticles enhance the radiosensitivity of VPA in glioblastoma. Transl Oncol 2017;10:229–40. doi: 10.1016/j.tranon.2016.12.011. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref15] 15. Liao G, Li R, Chen X et al. Sodium valproate prevents radiation-induced injury in hippocampal neurons via activation of the Nrf2/HO-1 pathway. Neuroscience 2016;331:40–51. doi: 10.1016/j.neuroscience.2016.06.019. [DOI] [PubMed] [Google Scholar]

[ref16] 16. Scott GA, McClelland LA, Fricke AF. Semaphorin 7a promotes spreading and dendricity in human melanocytes through beta1-integrins. J Invest Dermatol 2008;128:151–61. doi: 10.1038/sj.jid.5700974. [DOI] [PubMed] [Google Scholar]

[ref17] 17. Kumanogoh A, Kikutani H. Roles of the semaphorin family in immune regulation. Adv Immunol 2003;81:173–98. doi: 10.1016/s0065-2776(03)81005-2. [DOI] [PubMed] [Google Scholar]

[ref18] 18. Roth JM, Kohler D, Schneider M et al. Semaphorin 7A aggravates pulmonary inflammation during lung injury. PLoS One 2016;11:e0146930. doi: 10.1371/journal.pone.0146930. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref19] 19. Mizutani N, Nabe T, Yoshino S. Semaphorin 7A plays a critical role in IgE-mediated airway inflammation in mice. Eur J Pharmacol 2015;764:149–56. doi: 10.1016/j.ejphar.2015.07.004. [DOI] [PubMed] [Google Scholar]

[ref20] 20. Bussolino F, Giraudo E, Serini G. Class 3 semaphorin in angiogenesis and lymphangiogenesis. Chem Immunol Allergy 2014;99:71 –88. doi: 10.1159/000353315. [DOI] [PubMed] [Google Scholar]

[ref21] 21. Nakamura F, Kalb RG, Strittmatter SM. Molecular basis of semaphorin-mediated axon guidance. J Neurobiol 2000;44:219–29. doi: . [DOI] [PubMed] [Google Scholar]

[ref22] 22. Potiron V, Roche J. Class 3 semaphorin signaling: the end of a dogma. Sci STKE 2005;2005:pe24. doi: 10.1126/stke.2852005pe24. [DOI] [PubMed] [Google Scholar]

[ref23] 23. Yoshida Y. Semaphorin signaling in vertebrate neural circuit assembly. Front Mol Neurosci 2012;5:71. doi: 10.3389/fnmol.2012.00071. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref24] 24. Talhada D, Goncalves I, Reis Santos C et al. Transthyretin expression in the postischemic brain. PLoS One 2019;14:e0221555. doi: 10.1371/journal.pone.0221555. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref25] 25. Feng Z, Scott SP, Bussen W et al. Rad52 inactivation is synthetically lethal with BRCA2 deficiency. Proc Natl Acad Sci U S A 2011;108:686–91. doi: 10.1073/pnas.1010959107. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref26] 26. Liu G, Wang H, Zhang F et al. The effect of VPA on increasing radiosensitivity in osteosarcoma cells and primary-culture cells from chemical carcinogen-induced breast cancer in rats. Int J Mol Sci 2017;18. doi: 10.3390/ijms18051027. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref27] 27. Pugalendhi P, Manoharan S, Suresh K et al. Genistein and daidzein, in combination, protect cellular integrity during 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary carcinogenesis in Sprague-Dawley rats. Afr J Tradit Complement Altern Med 2011;8:91–7. doi: 10.4314/ajtcam.v8i2.63196. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref28] 28. Beresford MJ, Wilson GD, Makris A. Measuring proliferation in breast cancer: practicalities and applications. Breast Cancer Res 2006;8:216. doi: 10.1186/bcr1618. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref29] 29. Guerriero JL, Sotayo A, Ponichtera HE et al. Class IIa HDAC inhibition reduces breast tumours and metastases through anti-tumour macrophages. Nature 2017;543:428–32. doi: 10.1038/nature21409. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref30] 30. Dos Anjos Cassado A. F4/80 as a major macrophage marker: the case of the peritoneum and spleen. Results Probl Cell Differ 2017;62:161–79. doi: 10.1007/978-3-319-54090-0_7. [DOI] [PubMed] [Google Scholar]

[ref31] 31. Cole-Healy Z, Vergani P, Hunter K et al. The relationship between semaphorin 3C and microvessel density in the progression of breast and oral neoplasia. Exp Mol Pathol 2015;99:19–24. doi: 10.1016/j.yexmp.2015.03.041. [DOI] [PubMed] [Google Scholar]

[ref32] 32. Xue W, Warshawsky D. Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: a review. Toxicol Appl Pharmacol 2005;206:73–93. doi: 10.1016/j.taap.2004.11.006. [DOI] [PubMed] [Google Scholar]

[ref33] 33. Farrell G. Effects of disease on expression and regulation of CYPs. Mol Aspects Med 1999;20:55–70137. [PubMed] [Google Scholar]

[ref34] 34. Gallagher EP, Wienkers LC, Stapleton PL et al. Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin B1. Cancer Res 1994;54:101–8. [PubMed] [Google Scholar]

[ref35] 35. Gasiewicz TA, Henry EC, Collins LL. Expression and activity of aryl hydrocarbon receptors in development and cancer. Crit Rev Eukaryot Gene Expr 2008;18:279–321. doi: 10.1615/critreveukargeneexpr.v18.i4.10. [DOI] [PubMed] [Google Scholar]

[ref36] 36. Fitzpatrick FA. Inflammation, carcinogenesis and cancer. Int Immunopharmacol 2001;1:1651–67. doi: 10.1016/s1567-5769(01)00102-3. [DOI] [PubMed] [Google Scholar]

[ref37] 37. Wilson AS, Power BE, Molloy PL. DNA hypomethylation and human diseases. Biochim Biophys Acta 2007;1775:138–62. doi: 10.1016/j.bbcan.2006.08.007. [DOI] [PubMed] [Google Scholar]

[ref38] 38. Rizzolio S, Tamagnone L. Semaphorin signals on the road to cancer invasion and metastasis. Cell Adh Migr 2007;1:62–8. doi: 10.4161/cam.1.2.4570. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref39] 39. Malik MF, Satherley LK, Davies EL et al. Expression of Semaphorin 3C in breast cancer and its impact on adhesion and invasion of breast cancer cells. Anticancer Res 2016;36:1281–6. [PubMed] [Google Scholar]

[ref40] 40. Herman JG, Meadows GG. Increased class 3 semaphorin expression modulates the invasive and adhesive properties of prostate cancer cells. Int J Oncol 2007;30:1231–8. [PubMed] [Google Scholar]

[ref41] 41. Chen Y, Low TY, Choong LY et al. Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells. Proteomics 2007;7:2384–97. doi: 10.1002/pmic.200600968. [DOI] [PubMed] [Google Scholar]

[ref42] 42. Heeg S, Das KK, Reichert M et al. ETS-transcription factor ETV1 regulates stromal expansion and metastasis in pancreatic cancer. Gastroenterology 2016;151:540–553 e514. doi: 10.1053/j.gastro.2016.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ref43] 43. Tian X, Han Z, Zhu Q et al. Silencing of cadherin-17 enhances apoptosis and inhibits autophagy in colorectal cancer cells. Biomed Pharmacother 2018;108:331–7. doi: 10.1016/j.biopha.2018.09.020. [DOI] [PubMed] [Google Scholar]

[ref44] 44. Zeweil MM, Sadek KM, Taha NM et al. Graviola attenuates DMBA-induced breast cancer possibly through augmenting apoptosis and antioxidant pathway and downregulating estrogen receptors. Environ Sci Pollut Res Int 2019;26:15209–17. doi: 10.1007/s11356-019-04920-w. [DOI] [PubMed] [Google Scholar]

PERMALINK

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

Junxuan Peng

Zuchao Cai

Ruixue Zhao

Jiahao Chen

Guochao Liu

Chao Dong

David Lim

Zhihui Feng

Abstract

Graphical abstract

Introduction

Materials and methods

The establishment of the animal model

Tumor observation on rats

BrdU incorporation and HE staining

Immunohistochemistry staining

RNA-seq

Real-time quantitative reverse transcriptase PCR

Table 1.

Western blot analysis

Statistical analysis

Results

VPA can reduce DMBA-induced tumor formation in rats

Figure 1.

VPA can inhibit the proliferation of DMBA-induced tumor cells in rats

Figure 2.

VPA had a protective effect on normal tissues in DMBA-exposed rats

Figure 3.

Figure 3.

Figure 3.

The analysis of the potential genes associated with DMBA-induced tumorigenesis

Table 2.

VPA potentially inhibit the formation of DMBA-induced tumors by interfering with the expression of tumor-associated genes Sema3c

Figure 4.

Discussion

Conclusion

Conflict of interest statement

Supplementary Material

Acknowledgments

Contributor Information

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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