Background
Colon cancer is the malignant carcinoma and the third most common cancer worldwide leading to causes of deaths [
1]. The early stage of colon carcinoma can be treated by using surgical extraction, radiotherapy and drugs. Colon cancer or infectious agents can be as a danger molecule in our tissue, and then immune system cells respond to the damage and intent to eliminate the danger [
2,
3]. These immune cells produce pro-inflammatory cytokines IL1β, IL6 and TNFα to increase strength of the response against danger [
4]. Previous study has shown that the anti-inflammatory drugs are associated with the reduced incidence and the metastasis rat in the colon cancer [
5]. Inflammation is one of the main reasons and critical incidents for cancer progression and poor prognosis, particular in colon cancer [
6]. Thus the molecular mechanisms between colon cancer and inflammation demand for further study in detail which can provide the treatment strategies for colon cancer.
HMGB family, including HMGB1, HMGB2 and HMGB3, have been studied for the past decade as the nuclear components important for transcription and chromatin structure [
7]. HMGB1, also termed HMG1 and amphoterin, is most abundant member of HMGB family of the DNA-binding proteins [
8‐
10]. Normally, HMGB1 protein exist in cell nucleus; when it is released to the extracellular space upon the cell damage, it becomes an immune-inflammatory factor [
11‐
13]. In general, the stimulation of cells by cytokines, lipopolysaccharide (LPS) or hypoxia enables HMGB1 export from nucleus to cytoplasm or the extracellular space, and acetylation of HMGB1 has been suggested to regulate its intracellular shutting in cells [
14,
15]. Evidences have shown that the extracellular HMGB1 can be as a mediator of inflammation and tissue regeneration [
8,
16]. HMGB1 is closely related to many inflammatory diseases, such as ischemia of liver and kidney, hepatitis, arthritis, stroke, ischemia of liver and kidney, sepsis, and systemic lupus erythematosus [
17‐
19]. Yet, the link between HMGB1 and inflammation in colon cancer remains unclear, and then we have proposed the hypothesis that HMGB1 mediated the inflammation in colon cancer.
GPX (glutathione peroxidase) is the intracellular anti-oxidant enzyme responsible for reducing peroxides. GPX4, a member of the GPxs family, also called phospholipid hydroperoxide glutathione peroxidase (PHGPx), is considered one of the important antioxidant enzymes in the mammals [
20‐
22]. GPX4 is especial among the GPX family in that it has a unique affinity for membrane-bound substrates such as the phospholipids and cholesterol hydroperoxides [
23‐
25], directly reducing the membrane-bound phospholipid hydroperoxides in the situ and protecting against damage. Lipid hydroperoxides are implicated in variety of pathophysiological processes, including inflammation. Besides being able to repair lipid peroxides as the antioxidant enzyme, GPX4 can also regulate cytokine signaling [
26]. GPX4 is identified as a central regulator of the ferroptosis, a newly discovered the iron-dependent cell death and latest addition to list of the surprises [
27,
28]. However, just like HMGB1 as a mediator of inflammation, GPX4 can be also responsible for inflammation, the connections of HMGB1 and GPX4 are not currently known.
Here we report that HMGB1 mediates LPS-induced inflammation in colon cancer cells. LPS was used to mimic the danger and then stimulate SW480 and HCT116 colon cells. In order to explore the inflammation under LPS stimulated conditions, we detected the mRNA levels of pro-inflammatory cytokines IL1β, IL6 and TNFα. Our results revealed that HMGB1 can bind with GPX4, thus mediating the occurrence and development of inflammation induced by LPS. Our discoveries provide a novel molecular mechanism of inflammation in colon cancer cells and the therapeutic strategies for colon cancer.
Materials and methods
Plasmid construction and siRNA
cDNA encoding GPX4 was synthesized and cloned into the pcDNA3.1(+) expression vector (Invitrogen) using the segment and fusion PCR. Meanwhile, To silence the expression of HMGB1 in SW480 and HCT116 cells, siHMGB1 was purchased from Santa Cruze. After seeding into the 6-well plates, the siHMGB1 or pcDNA3.1-GPX4 were transfected into SW480 and HCT116 cells utilizing Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). The protein levels of HMGB1 and GPX4 were detected after transfection for 48 h by western blot.
Cell culture and treatment
SW480 and HCT116 colon cancer cells were maintained in School of Biological Science and Technology, Chengdu Medical College. The cells were cultured in the Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovin serum (FBS) and then transfected with the jetPRIME (#114-15, SA, France), thus incubated in the 5% CO2 incubator at 37 °C. For treatment, after transfetion, the cells were pretreated utilizing various does of GL (glycyrrhizin) (Sigma, St. Louis, MO, USA) (1 mmol/L) for 12 h and stimulated with 1 μg/ml LPS for 6 h. Then cells were harvested and analyzed using the appropriate antibodies. All experiments were repeated for three times.
Quantitative real-time PCR (qRT-PCR) assay
For quantitative real-time PCR, the cells under various treatment were collected by using TRIzol (Invitrogen) to extract the total RNA. And then The cDNA was synthesized by utilizing the SuperScript II First Strand Synthesis System (Invitrogen). The qRT-PCR was performed to quantify the IL-1β, IL-6 and TNF-α transcript levels by using the specific primers. The β-actin, housekeeping gene, was an internal standard to calculate the target gene relative expression using the 2 − ΔΔCT formula. qRT-PCR was performed by the quantitative real-time PCR system (BioRad, Hercules, CA, USA), with the first denaturation step, followed with the 40 cycles containing denaturaion, annealing and extension.
Immunoprecipitation (IP) and western blot
For IP assay, the cells were lysed by using the protein extraction buffers (PP1801, Bioteke, China) containing the protease inhibitor (04693116001, Roche, Switzerland) after transfected with siHMGB1 and pcDNA3.1-GPX4 for 48 h. The extracted protein was immunoprecipitated with the specific antibody and protein A + G agarose beads (P2012, Beyotime, China). For western blot, equal amounts of proteins were separated with SDS-PAGE and subsequently transferred to the PVDF membrane, then detected using primary antibody and the horseradish peroxidase-conjugated secondary antibodies. After that, the special immunoreactive bands were detected with chemiluminescence western blot detection system (WBKLS0100, Millipore, USA).
ROS detection by fluorescence microscope
To detect the intracellular ROS levels in SW480 and HCT116 cells, Reactive Oxygen Species Assay Kit (S0033, Beyotime, China) was used. The cells were seeded in a 24-well culture plate at a density of 2 × 105 cells/well. The reagents freely enter into the cells and then become highly fluorescent after being oxidized by the ROS. LPS, siHMGB1 and pcDNA3.1-gpx4 administration, the medium of culture plate was discarded, and then cells were washed using PBS (phosphate-buffered saline). Subsequently, the colon cells were incubated with 10 μM reagents for 20 min at 37 °C, and the results were analyzed by fluorescence microscope.
GPX4 activity assay
GPX4 activity assay was performed using the Glutathione Peroxidase Activity Colorimetric Assay Kit (Bioversion, catalog: #K762-100) according to manufacturer’s instructions. Colon cancer cells on ice in 0.2 ml cold assay buffer; Centrifuge at 10,000g for 15 min at 4 °C; Collect the supernatant for assay and store on ice. Serum can be tested directly. We suggest testing several doses of your sample to make sure the readings are within the standard curve range. The cellular extracts of colon cancer cells treated with the DMSO or the indicated concentrations of LPS for 6 h were then prepared according to manufacturer’s instructions. GPX activity assay was assessed via measuring changes in absorbance at 340 nm to the NADPH standard curve.
Immunohistochemistry
Colon cancer tissues were sliced at the thickness of 4 μm, and then sections were placed on the silane-coated slides and deparaffifinized. Antigen retrieval based on heat, the endogenous peroxidase block with 3% hydrogen peroxide, and then blocking were performed using normal sera. The used primary antibodies were HMGB1, GPX4, and p-p65. Specimens with the antibodies were incubated for overnight at 4 °C. The visual color was performed by using the diaminobenzidine (DAB; Nichirei Bioscience, Japan).
Statistical analysis
The whole data were repeated at least three separate times, and were expressed as the mean ± SD with the GraphPad Prism 7.0 software. The statistical analyses were analyzed using the SPSS version 17.0. For the comparison, differences were determined with the Student’s test among the experimental groups. Statistical significance was considered as the Values of P < 0.05.
Discussion
Colon cancer is malignant tumor leading causes of deaths and a challenge to the clinicians throughout the world [
1]. Evidences have shown that inflammation is a critical reason for colon cancer progression and poor prognosis [
6]. Thus the clarification of this mechanisms for colon cancer treatment is extremely urgent. In this study, our immunoprecipitation results showed that HMGB1 can interact with GPX4 in both colon cancer cells, thus leading to ROS accumulation and inflammation stimulated by LPS, identifying the novel mechanism for colon cancer therapy.
Studies have shown that LPS can induce inflammation and oxidative stress [
34‐
36]. To verify LPS-stimulated cell models, we investigated the effects of LPS on the mRNA level of pro-inflammatory cytokines
IL1β,
IL6 and
TNFα and ROS levels in SW480 and HCT116 cells, and then our data determine this effect. As a inflammation mediator and a biomarker and drug target, HMGB1 is currently related to many inflammatory diseases [
37]. In this study, the results showed that siHMGB1 suppress inflammatory gens mRNA and ROS levels, in contrast, elevated the GSH level, showing that HMGB1 is involved in inflammation and oxidative stress. Moreover, HMGB1 and ROS are associated with NF-kB pathway, when siHMGB1 NAC were introduced, p-IKBα and p-p65 protein expression were effectively decreased, thus inhibiting the NF-kB-dependent gens transcription. Since NF-kB as mediator effect on inflammatory response [
29,
38], above results further demonstrated that HMGB1 mediates LPS-induced inflammation in colon cancer cells.
On the other hand, besides being as a antioxidant enzyme, the current studies have shown that the activation of GPX4 helps to promote the inflammation resolution by eliminating the reactive oxygen species [
39,
40]. Current studies have shown both HMGB1 and GPX4 contribute to inflammation, then we investigated the linkage of HMGB1 and GPX4, and the results showed that HMGB1 could interact with GPX4 and negatively regulated the GPX4 activity, while lacking of LPS-stimulated made HMGB1 fail to interact with GPX4. Under the LPS stimulated conditions, HMGB1 can export from nucleus to cytoplasm and Glycyrrhizic acid, the inhibitor of HMGB1, can block its translocation. Meanwhile, the levels of acetylated HMGB1 was significantly elevated, verifying that acetylated HMGB1 is responsible for interaction with GPX4. Thus our discoveries provided a novel mechanism for inflammation mediated by HMGB1.
Subsequently, by utilizing 50 pairs of colon tumor tissues, we analyzed the expression and correlation about HMGB1, GPX4 and p-p65, and then evaluated their correlation in colon cancer. The present study showed HMGB1 expression were positively correlated with GPX4 and p-p65, these clinical findings suggest the therapeutic targets for colon cancer.
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