Background

It is reported that laryngeal cancer has a higher incidence in developed countries and as is known to all that in the head and neck laryngeal cancer is one of the important malignant cancers (Jemal et al., 2011). There are some conventional ways such as surgery, chemotherapy and radiotherapy to treat laryngeal cancer. But there are poor curative effect and rate, and it has requirement to improve significantly. There are many chemotherapeutic drugs that can be available to treat laryngeal cancer, but there should have a way to cure laryngeal cancer which is highly effective and less toxic.

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a naturally-occurring phytochemical and the major pungent constituent of hot chili peppers of the genus Capsicum (family Solanaceae), which are extensively used as food additives. In different human cancer models, some studies showed capsaicin was a chemopreventive and chemotherapeutic agent (Hail et al., 2002; Surh, 2002; Ito et al., 2004; Mori et al., 2006; Sánchez et al., 2006; Wu et al., 2006). Through a p53-dependent mechanism, capsaicin induced apoptosis in leukemia cells (Mori et al., 2006). Capsaicin elevated caspase-3 activation and Ca²⁺ productions and intracellular reactive oxygen species (ROS), and in this way capsaicin triggered apoptosis in human esophagus epidermoid carcinoma cells (Wu et al., 2006). In a way of p53-independent pathway, the apoptosis was connected with capsaicin-affected prostate cancer cells (Sánchez et al., 2006). The adenosine monophosphate-activated protein kinase activator elevated capsaicin-induced apoptosis by cotreatment in colorectal cancer cells (Kim et al., 2007). But in rats in vivo azoxymethane-induced aberrant cryptic foci formation was suppressed by capsaicin treatment (Yoshitani et al., 2001).

The phosphatidylinositol-3 kinase/serine-threonine kinase, which is the signaling pathway PI3K/Akt, influences cell growth and proliferation, which is include protein synthesis, glucose metabolism and the activation of anti-apoptotic mechanisms (Chang et al., 2003; Fresno et al., 2004). In many malignancies, the PI3K/Akt pathway is found to be abnormal activated. But as is known to all, it is important that PI3K which is tyrosine kinase-mediated is activated. Such as, phosphorylated tyrosine kinase which interacts with the p85 subunit leads to the activation of PI3K, at the same time PI3K which is mutated Ras-binding to causes the PI3K activation.

Apart from this, an important role is acted by somatic mutations. Such as the ability of PTEN to switch off the PI3K pathway in the PTEN tumor-suppressor gene may be disrupted by mutation. Nowadays in about 30% of epithelial tumors, there found a PIK3CA mutation. In the process of the cancers, such as breast, colon, pancreatic, lung and ovarian cancer or other cancers, somatic mutations and abnormal activation of PI3K improve abnormal cell proliferation and growth collectively (Oganesian et al., 1997; Chang et al., 2003; Watanabe et al., 2009).

In this study, in vivo and in vitro we proposed to observe the application of capsaicin to treat laryngeal cancer, and study the probably capsaicin tumoricidal mechanism. At the same time, we discussed the prevention of laryngeal cancer and effect on the laryngeal cancer treatment.

1 Results

1.1 Following capsaicin treatment laryngeal cancer cell growth marked inhibited

The treated and negative control groups noted in cell growth obvious differences that was in the (24, 48 and 72 h) time-dependent and Hep-2 cells dose-dependent (100, 200 and 300 µM) treatment with capsaicin (P<0.05) (Figure 1A). At the 24, 48 and 72 h time points, respectively, the Hep-2 cell proliferation inhibition percentage was 15.9, 21.8 and 19.1%, that was in the group treated with 100 µM capsaicin. At the same time, respectively, at 24, 48 and 72 h the percentage of cell proliferation inhibition was 23.2, 25.8 and 33.8%, that was in the group treated with 200 µm capsaicin. Respectively, the percentage of cell proliferation inhibition at 24, 48 and 72 h were 28.9, 48.6 and 64.3%, that was in the group treated with 300 µm capsaicin (Figure 1B). It showed the capsaicin inhibited the Hep-2 laryngeal cancer cells proliferation which was highly dose dependent.

Figure 1 Capsaicin influences Hep-2 cell proliferation Note: A: Hep-2 cells were treated with capsaicin at concentrations of 0, 100, 200 and 300 µM, for 24, 48 and 72 h. The proliferation of the laryngeal cancer cells was detected by the CCK-8 method. The absorbance values of the wells are expressed as means ± SD, P<0.05 vs. negative control (0 µM). B: Relationship between the percentage of laryngeal cancer cell proliferation inhibition and different concentrations of capsaicin

1.2 Following capsaicin treatment laryngeal cancer Hep-2 cell apoptosis inducted

After 0, 100, 200 or 300 µM capsaicin 48 h treatment, by Annexin V-PI double staining the analysis of Hep-2 apoptosis showed respectively the apoptosis rate was 0.4±0.35, 2.5±1.42, 19.2±2.28 and 20.8±5.37%. At the same time, in the rate of apoptosis, the 100 and 0 µM was no statistically significant difference. And when compared between the 0 µM and the 200, 300 µM groups, the apoptosis rates were significant increases (P<0.05). This study result showed that Hep-2 cells apoptosis were induced in a 100 µM capsaicin concentration, and the apoptotic cells proportion were increased in a concentration of 150 µM and that showed an apoptosis rate >20% was displayed (Figure 2). The result showed that after the treatment with capsaicin, the apoptosis proportion of cells was dose-dependent (Figure 2).

Figure 2 FACS analysis of the effect of capsaicin on laryngeal cancer cell apoptosis Note: Hep-2 cells were treated with 0, 100, 200, 300 µM of capsaicin for 48 h, and examined following Annexin V-PI double staining for the detection of the apoptotic rate. P<0.05 vs. negative control (0 µM)

1.3 Following treatment with capsaicin protein expression of PI3K/Akt in Hep-2 cells obviously reduced

The key signaling proteins expression of the pathway PI3K/Akt and the downstream signaling proteins were examined and that were connected with proliferation and apoptosis of the cancer cell. The protein expression was all highly increased, which included PI3K class III, p-Akt, PI3K p110α, GSK3-β and p-c-Raf and that in negative control and the untreated control groups. When in the untreated groups compared with these levels, the expression of the protein was markedly decreased by Hep-2 cells treatment with capsaicin, which included the PI3K p110α, PI3K p85, p-Akt, Akt, p-GSK3-β, and GSK3-β the protein and the signaling proteins was capsaicin, which included the PI3K p110α, PI3K p85, p-Akt, Akt, p-GSK3-β, and GSK3-β the protein and the signaling proteins was capsaicin dosedependent according to the decreased expression levels (Figure 3).

Figure 3 Western blot analysis of the expression of PI3K/Akt signaling pathway proteins and downstream proteins in Hep-2 cells after capsaicin treatment Note: Hep-2 cells were treated with 0, 100, 200 and 300 µM of capsaicin following which the expression of key proteins of the PI3K/Akt pathway and downstream proteins were analyzed by western blot assay. Expression of GAPDH served as an internal reference control

1.4 In a BALB/c nude mouse model capsaicin tumoricidal effects

In animal model experiments, the capsaicin tumoricidal effect was further studied. Only conventional feed was received by the control mouse group. Subsequently tumors of a large size were developed in these mice. 2275.3±263.3mm³ was the average volume of the tumors at week 8. By contrast, in the capsaicin treatment group and the capsaicin pretreatment group, the mean volumes of the tumors respectively were 1697.2±152.3mm³ and 1279.2±241.5 mm³ (P<0.05). This result showed that tumor growth was significantly inhibited by the capsaicin (P<0.05), and that before tumor transplantation, administration of capsaicin was not less valid than after tumor transplantation, administration of capsaicin was established (P<0.05).

1.5 After capsaicin treatment in the transplanted tumors expression of PI3K/AKT and downstream signaling proteins significantly reduced

In the transplanted tumors the proteins expression that were connected to the pathway PI3K/AKT and signaling pathways of the downstream was analyzed and showed reduced expression of PI3K p110α, PI3K p85, p-Akt and p-GSK3-β signaling proteins were associated with increasing concentrations of capsaicin. The treatment group was the lower signaling protein expression and the pretreatment group which was the fewest the expression of the signaling protein. As expected, the signaling proteins the highest expression was the control group (Figure 4).

Figure 4 Hep-2 xenograft tumor model; capsaicin inhibition of tumor growth via the PI3K/Akt pathway Note: Eight weeks after tumor implantation, expression of proteins of the PI3K/Akt pathway in tumor tissue was analyzed by western blot analysis as described in Materials and methods. Expression of GAPDH served as an internal reference control. From left to right, the protective group (or the prevention group), the capsaicin treatment group and the control group

1.6 In the heart, liver and kidney of capsaicin-treated nude mice no harmful side effects are noted

With H&E specimens were sectioned and stained, that were obtained in the nude mice from the heart, kidney and liver. In the three groups of nude mice no adverse effects on the heart, liver or kidney were exerted by treatment with capsaicin through pathological evaluation. Any of the tissue specimens noted no such as structural degeneration or tissue necrosis tissue damage. In the experimental mice, the study showed that in any of the organs no toxic side effects were provoked by the feed with 0.5% (w/w) capsaicin supplementation (Figure 5).

Figure 5 Hematoxylin and eosin (H&E) staining of the heart, liver and kidney from capsaicin-treated nude mice Note: The heart, liver and kidney sections from the three groups of nude mice (from left to right: the protective group (or the prevention group), the treatment group and the control group) were stained with H&E to observe and assess cell morphology and tissue structure (×400)

2 Discussion

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a naturally-occurring phytochemical and the major pungent constituent of hot chili peppers. In vivo and in vitro, many previous studies have indicated that by inducing cell apoptosis and cycle arrest a lot of tumor cell lines were inhibited significantly by capsaicin (Hail et al., 2002; Surh, 2002; Ito et al., 2004; Mori et al., 2006; Sánchez et al., 2006; Wu et al., 2006). But, the effect of capsaicin in Hep-2 cells was not clear largely. The study showed that proliferation of Hep-2 cells was inhibited by capsaicin significantly and the tumor inhibition rate was respectively 28.9, 48.6 and 64.3% noted at 24, 48 and 72 h, which was at an optimal concentration 300 µM. Induction of apoptosis and alteration of the proliferation of cancer cells were involved in the inhibitory mechanism. The study showed an apoptotic effect was induced at a dose of 200 µM of capsaicin and in a capsaicin dose-dependent manner, the apoptotic rate was improved. Thoennissen et al. (2010) showed apoptosis was induced by with 50-200 µM of capsaicin treatment of breast cancer cells for 24-72 h.

Receptor tyrosine kinase signaling pathways is related with the PI3K/Akt pathway. phosphorylated Akt (p-Akt) and downstream target proteins Akt and PI3K can be inappropriately activated by mutational activation of pathway components, and activation and phosphorylation of mdm2, mTORGSK3, Bad, and the caspase family members can be subsequently resulted in, and tumor cell proliferation, growth, promotion of cellular invasion, tumor metastasis, suppression of apoptosis and angiogenesis collectively can be occurred (Brognard et al., 2001). The development of head and neck squamous cell carcinomas and tongue carcinoma can be found to be closely in particular related with abnormal phosphorylation of Akt and abnormalities in the signaling pathway PI3K/Akt (Oganesian et al., 1997; Saiki, 2011).

Both before and after treatment with capsaicin, the differential expression of pathway-specific signaling proteins PI3K/Akt and their cognate downstream proteins were analyzed, which in order to further clear the probable connection in laryngeal cancer between capsaicin-induced apoptosis and the signaling pathway PI3K/Akt. As in the untreated cells compared with levels, following treatment with capsaicin noted decreased expression of PI3K p110α, PI3K p85, p-GSK3-β, and p-Akt in Hep-2 cells markedly. The study shows that the signaling pathway PI3K/Akt is closely associated with laryngeal cancer cell proliferation inhibition and apoptosis induction. The results are consistant with those that been found in prostate or esophagus cancer (Wu et al., 2006; Saiki, 2011).

Previous studies (Mori et al., 2006; Sánchez et al., 2006) indicated that the solid tumors occurrence and development is inhibited by capsaicin. The studies of colon carcinoma showed that the improvement in the tumor cell membrane permeability, caspase-3 activation was partly due to the antitumor mechanism of capsaicin, and in the end induce cancer cell apoptosis (Borner, 2003; Kim et al., 2004; Gil and Kang, 2008; Kim et al., 2009; Oyagbemi et al., 2010). Over a period of 10 weeks, the laryngeal cancer cells apoptosis was produced, after the animals with breast cancer were fed diets containing 1% (w/w) capsaicin (Thoennissen et al., 2010) and a pathway which is known to control antioxidant signaling pathways which called Nrf2 was activated. Ip et al. (2012) indicated that 2-hydroxy estrone which is the anti-proliferative agent could be produced by triggering the immune response of fed with capsaicin mice. The mice experiments showed that the growth of laryngeal cancer could be effectively inhibited by capsaicin and when compared with the control mice, laryngeal cancer growth in nude mice could be significantly decreased by capsaicin intervention. The study shows capsaicin prevents the solid tumors development and occurrence effectively, which is due to that the graft tumor growth rate in the pretreatment group was not more rapid than which noted in the treated mouse group. The result also shows that for the treatment group and prevention group of laryngeal cancer capsaicin can be regarded as an alternative therapeutic way.

The result showed that be compared with the levels in the control group which was not cured with capsaicin, the p-GSK3-β, p-Akt, PI3K p85 and PI3K p110α expression levels were downregulated obviously, which was consistent with the results that were obtained from in the treated and pretreated experiment mice in vitro experiments. The experiment shows that in the process of the laryngeal cancer, the PI3K/Akt pathway abnormal activation may do an important role. In the transplanted tumors, the mice experiment also indicated that in the signaling pathway PI3K/Akt, protein expression was downregulated by capsaicin.

Chou et al. (2009) indicated that MCF-7 breast cancer cell apoptosis only be induced by capsaicin, when mammary epithelial homologous CF10A cells and breast cancer MCF10CA1a cells were treated by capsaicin, and this experiment showed capsaicin was without risk to non-tumor cells. At the same way, our result also indicated that in nude mice growth inhibition was caused by capsaicin in the transplanted tumors, and it showed that there was low cytotoxicity to normal cells and any degenerative harm that produced to the heart, liver, kidney and other organs has been provoked.

3 Materials and methods

3.1 Cell cultures and reagents

Hep-2 laryngeal cancer cell line cell line was maintained at 37˚C in a humidified atmosphere with 5% CO₂ and in RPMI-1640 which contained 100 mg/ml streptomycin, 100 U/ml penicillin and 10% fetal bovine serum (Hyclone, UT, USA), which was bought in the Shanghai Cell Collection in China. Beijing HFK Bioscience Co., Ltd. (China) provided specific pathogen-free which is called SPF female BALB/c nude mice, that were 18-20 g weight and 4 weeks age. Sigma-Aldrich, St. Louis, MO, USA provided capsaicin.

3.2 Cell treatment

Dimethyl sulphoxide (DMSO) dissolved capsaicin and capsaicin got a stock 1 M concentration, and at last capsaicin was diluted to get the concentrations 100, 200 and 300 µM, which used for the experiments. Capsaicin preparations the final concentration of DMSO was less than 0.5%. In complete medium Hep-2 cells were cultured alone, and the aim was to show Hep-2 cells did not be affect by the final concentration of DMSO, that were used as the untreated control, and at the same time the negative control group (0 µM) was DMSO (<0.5%).

3.3 Cell proliferation and cytotoxicity assay

96-well plates seeded Hep-2 cells in complete culture medium at a density of 1×10³/ml. And per concentration of capsaicin replicated in 6 wells with capsaicin at 0, 100, 200 or 300 µM. Blank control was the other contained culture medium alone 6 wells. CCK-8 reagent (Dongji, Japan) 10 µl treated Hep-2 cells and Hep-2 cells were incubated at 37˚C for 1 h after 0, 24, 48 and 72 h. On the basis of the blank control wells, zero set to the automatic microtitre plate reader. A wavelength of 450 nm was the absorbance (A) of the wells. In light of the formula, which was the inhibition of cell proliferation (%) = (1- the experimental group average absorbance (A) / the control group average absorbance (A))×100%, it got the cell proliferation inhibition the percentage.

3.4 Using annexin V‑FITC and propidium iodide flow cytometric analysis of apoptotic cells (PI)

6-well plates seeded Hep-2 cells. 0, 100, 200 and 300 µM of capsaicin contained in complete culture medium. And an untreated control group was seeded with culture medium separate wells alone. Each group was seeded in triplicate. Hep-2 cells were gained by centrifugation, the cells were resuspended in binding buffer, and at room temperature for 15 min the cells were incubated with 5 µl of PI and 5 µl Annexin V-FITC (Multi Sciences, China). Flow cytometric analysis determined apoptosis, which used a FACSCanto™II spectrophotometer (BD Biosciences, San Jose, CA, USA). The vertical axis set PI staining and horizontal axis set V staining in the flow cytometric dot plot. Apoptotic cells early were located in the flow cytometric dot plot lower right quadrant and dual negative and normal cells in the lower left quadrant. And necrotic or apoptotic cells were posited in the right upper quadrant and mechanically damaged cells were located in the left upper quadrant.

3.5 Western blot analysis

RIPA buffer lysed Hep-2 cells and extracted total laryngeal cancer cellular protein, when different concentrations of capsaicin treated the cells. The BCA quantitative method determined protein concentration, and SDS-PAGE resolved each protein sample 30 µg and the nitrocellulose membrane transferred the protein bands. In the 5% skimmed milk proteins presence, it blocked the membrane for 1 h after protein transfer, and the primary antibodies (Cell signaling Technology, Inc., Beverly, MA, USA) that been targeted against PI3K p85, p-Akt, PI3K p110α, p-GSK3-β, GSK3-β, Akt and GAPDH which regarded as an internal reference housekeeping protein incubated at 4˚C overnight. The next day, a secondary antibody incubated the blots, and an enhanced chemiluminescence (ECL) assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA) visualized specific protein bands.

3.6 Animal selection and breeding

Beijing HFK Bioscience Co., Ltd. (China) provided SPF which is called specific pathogen-free female BALB/c nude mice, that were18-20 g weight and 4 weeks age. SPF conditions carried out all of the animal experimentation and husbandry practices. Conventional feed provided the animals for 1 week before the experiment. At a 0.5% (w/w) ratio the conventional feed was incorporated into capsaicin after this adaptive feeding. The Wuhan University Animal Care and Use Local Ethics and Committee, China approved all the study.

3.7 Tumor-bearing animal models in BALB/c nude mice established

The study randomly assigned female BALB/c mice twenty-four to three groups: n=8 mice/group, a control group, a treatment group and a pretreatment group. A confluence of 80-90% cultured the Hep-2 cells, which were harvested by trypsin treatment and the cells were in plasma resuspended after centrifugation. When into the right side of the animals a 200 µl cell suspension was injected in plasma subcutaneously, a density of 1×10⁷ cells/ml adoptively transferred the Hep-2 cells. Before transfer adoptive Hep-2 cell, the conventional feed received the pretreatment group, which supplied by capsaicin at a 0.5% (w/w) ratio for 2 weeks. At the same time, after transfer with the Hep-2 cells, with capsaicin at a 0.5% (w/w) ratio of capsaicin the conventional diet supplemented received the pretreatment group. Until the endpoint at week 8, the capsaicin supplemented feed continued to the animals. In order to using the formula: V = 1/2ab² to compute the volume for statistical analysis, the study measured the long diameter (a) and the short diameter (b), after the Hep-2 cells adoptive transfer into the animals and at the end of each week. It should sacrifice the animals after eight weeks and collect the specimens including kidney, liver and heart.

3.8 Expression assay and protein extraction

It should from the sacrificed mice cut tumor specimens into sized pieces regularly and use a tissue homogenizer to mince the tissues. After this preparation, RIPA buffer lysed the cells and the BCA method quantified the extracted proteins. The specific protein expression of Akt, GSK3-β, PI3K and was detected by western blot assay.

3.9 The heart, liver and kidney tissue sections morphological observation

10% formalin fixed heart, kidney and liver biopsy specimens which from the nude mice were obtained and were dehydrated, embedded in wax, and last were into 5-to 8-µm specimens sectioned pasted specimens into slides. The next step, for final histological evaluation, de-waxed the specimens and hematoxylin and eosin (H&E) stained the specimens under a standard light microscope.

3.10 Statistical analysis

The software package, version 16.0 SPSS statistical assessed all statistical analyses for (SPSS Inc., Chicago, IL, USA) Microsoft Windows. Means ± standard deviation (SD) expressed results. In multiple groups differences were by ANOVA compared. A statistically significant result considered P<0.05.

4 Conclusion

In conclusion, the experiments indicated that laryngeal cancer proliferation was inhibited significantly by capsaicin and apoptosis was induced by capsaicin. Our opinions are that the regulation of the signaling pathway PI3K/Akt and the expression of the dowstream protein are likely associated with the observed effects of capsaicin. For laryngeal cancer, capsaicin is thought to be a probably curative and preventive candidate, which due to the non-toxic, natural antitumor, effective properties. It requires further study to explore the underlying mechanisms that how at the molecular level the laryngeal cancer is suppressed by capsaicin, and for the clinical utility and tumoricidal of capsaicin, it provides a theoretical basis.

Authors’ Contributions

Chenggang Mao and Xiaochun Zhou wrote and translated the manuscript. Zezhang Tao read and approved the final manuscript. Yidao Jiang, Haitao Lu and LijiaWan collected materials. All authors read and approved the final manuscript.

Acknowledgements

The study was supported by grants from the Science and Technology Program of Jingzhou City (nos. 2017038 and 2017044).

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