|Year : 2015 | Volume
| Issue : 7 | Page : 172-175
Receptor for activated protein kinase C 1 suppresses gastric tumor progression through nuclear factor-kB pathway
X Yong-zheng, M Wan-li, M Ji-ming, R Xue-qun
Department of General Surgery, Huaihe Hospital of Henan University, Kaifeng 475000, Henan Province, PR China
|Date of Web Publication||20-Jul-2016|
Department of General Surgery, Huaihe Hospital of Henan University, Kaifeng 475000, Henan Province
Source of Support: None, Conflict of Interest: None
OBJECTIVE: Nuclear factor-kB (NF-kB) activity is crucial for survival and proliferation of many kinds of malignancies, including gastric cancer (GC). The receptor for activated protein kinase C 1 (RACK1) is known to regulate tumor development, whereas the underlined mechanism has not been described clearly. MATERIALS AND METHODS: We analyzed expression of RACK1 in paired human GC samples by both real-time polymerase chain reaction (PCR) and western blot. Effects of RACK inhibition with small interfering RNA or its overexpression in cultured GC cell lines were evaluated in cell viabilities. NF-kB signaling was investigated using luciferase reporter assay and real-time PCR. RESULTS: RACK1 was significantly decreased in GC samples. Knockdown of RACK elevated GC cell viabilities, whereas overexpression of RACK1 suppressed tumorigenesis of GC cells. Importantly, NF-kB signaling was enhanced after RACK1 expression was inhibited, suggesting the negative regulation of the pro-oncogenic NF-kB activity by RACK1 might contribute to its tumor suppressor role in GC cells. CONCLUSION: Our results support that RACK1 suppresses gastric tumor progression through the NF-kB signaling pathway.
Keywords: Gastric cancer, nuclear factor-kB signaling, receptor for activated protein kinase C 1, tumor progression
|How to cite this article:|
Yong-zheng X, Wan-li M, Ji-ming M, Xue-qun R. Receptor for activated protein kinase C 1 suppresses gastric tumor progression through nuclear factor-kB pathway. Indian J Cancer 2015;52, Suppl S3:172-5
|How to cite this URL:|
Yong-zheng X, Wan-li M, Ji-ming M, Xue-qun R. Receptor for activated protein kinase C 1 suppresses gastric tumor progression through nuclear factor-kB pathway. Indian J Cancer [serial online] 2015 [cited 2019 Aug 22];52, Suppl S3:172-5. Available from: http://www.indianjcancer.com/text.asp?2015/52/7/172/186572
| » Introduction|| |
Gastric cancer (GC) now ranks the third leading cause of cancer-related mortality worldwide.  Although the incidence of GC has declined in the Western countries over the last decades, it is still common in Eastern Asia. ,, The invasion and metastasis of GC represent the major reasons for its poor prognosis.  Most GC patients have advanced or metastatic diseases at the time of diagnosis.  Hence, the median survival time for these patients is often <12 months.  Recently, efforts on researching on GC have added multiple new insights into the pathogenesis of this malignancy.  However, there is a long way to go for evaluation of more and efficient strategies for GC treatment and early diagnosis.
Nuclear factor-kB (NF-kB), a potential sign for inflammation, has been shown to be involved in the progression of various types of cancers. ,, It was originally identified as a heterodimeric transcription factor in the nuclei of mature B-lymphocytes and could regulate gene expression associated with inflammatory and immune responses. , Recent findings have indicated that NF-kB and the signaling pathways that are involved in its activation are also important for tumor development and progression, especially significantly associated with the oncogenic processes leading from inflammation to carcinogenesis.  Since Helicobacter pylori infection plays a role in gastric neoplastic transformation by causing chronic gastritis at an early stage of GC pathogenesis, the role of NF-kB signaling was intensively discussed previously. ,
Receptor of activated protein C kinase 1 (RACK1), also named as GNB2 L1, is a member of the intracellular receptors for activated protein kinase C. It is highly conserved and crucial scaffold protein, involved in the progression of various cancers by regulating multiple crucial cellular processes including cell proliferation, apoptosis, and migration. ,,, In GC research, Chen et al. recently reported that loss of RACK resulted in enhance of GC cell metastasis, via promoting the autocrine of interleukin (IL)-8 in vitro and in vivo.  Besides, Deng et al. previously had shown that RACK1 could suppress the gastric tumorigenesis by negatively regulating Wnt signaling pathway through stabilizing the β-catenin destruction complex and act as a tumor suppressor in GC cells.  However, whether RACK1 plays a tumor-suppressive role in GC cells through other kinds of mechanisms is still unclear.
| » Materials and Methods|| |
Cell culture and transfection
GC cell lines SGC-7901 was cultured in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 10 U/mL penicillin G, and 10 mg/mL streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 . SGC-7901 was transfected with Lipofectamine 2000 reagent Invitrogen (Shanghai, China).
Primary gastric cancer samples
Primary tissues were collected from patients who received surgery for GC at Huaihe Hospital of Henan University. All patients had given informed consent. Dissected samples were frozen immediately after surgery and stored at − 80°C until needed.
Plasmids and short interference RNAs
The open reading frame of human RACK1 complementary DNA was cloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA). The RACK1 small interfering RNAs (siRNAs) and control siRNAs were purchased from GenePharma (Shanghai, China).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
Sgc-7901 cells were plated in 96-well plates as 2000 cells/well. Twenty-four hours after transient transfection, cells were cultured continually. At 24, 48, 72, and 96 h, 10 μl of 0.5 Mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) was added to each well. Cells were incubated at 37°c for 4 h; the medium removed, and precipitated formazan dissolved in 100 μl dimethyl sulfoxide. After shaking for 30 min, absorbance was detected at od490.
Real-time polymerase chain reaction
RNA was extracted from the tissues of GC patients or transfected cells. Real-time polymerase chain reaction (PCR) was performed on Applied Biosystems Step Two Real-Time PCR System using the comparative threshold cycle quantization method. Real-time Master Mix (Toyobo, Osaka, Japan) was used to detect and quantify the expression level of target gene. Glyceraldehyde-3-phosphate dehydrogenase was as internal control.
Luciferase reporter assay
Cells were plated at a subconfluent density and cotransfected with 0.2 μg of the reporter plasmid, 0.5 μg of expression vectors, and 0.1 μg of β-galactosidase as an internal control for transfection efficiency. Cell lysates were prepared 24 h after transfection, and the reporter activity was measured using the luciferase reporter assay system (Promega, Madison, WI, USA). Transfections were performed in triplicate and repeated 3 times independently.
Data expressed as the mean ± standard deviation were analyzed by SAS version 9.2 using independent t-test and paired t-test. P < 0.05 was considered statistically significant.
| » Results|| |
Expression of receptor for activated protein kinase C 1 is reduced in gastric cancer samples
We already knew that RACK1 was decreased in GC tissues from previous reports. , In the present study, we confirmed this conclusion in our systems. Fifty pairs of GC tissues matched with adjacent normal gastric tissues were collected from clinic. Real-time PCR results [Figure 1]a illustrated a significant downregulation of RACK1 mRNA expression in these tested GC samples (P < 0.01). Afterward, we also measured the protein expression of RACK1 in 20 pairs of GC samples randomly selected from the above GC samples by western blot. The results revealed that the protein levels of RACK1 were also significantly reduced in these selected GC samples [Figure 1]b, P < 0.01]. Hence, through these initial studies, we concluded that RACK1 was indeed decreased in GC samples, which reflected a potential tumor suppressor role in GC tumorigenesis.
|Figure 1: The expression of receptor for activated protein kinase C 1 was downregulated in gastric cancer. (a) Relative expression of receptor for activated protein kinase C 1 mRNA in 50 paired gastric normal tissues and cancer tissues. (b) Receptor for activated protein kinase C 1 protein expression are shown as box plots, with the horizontal lines representing the median; the bottom and top of the boxes representing the 25th and 75th percentiles, and the vertical bars representing the range of data. We compared the expression of receptor for activated protein kinase C 1 in normal tissues and cancer tissues using the t-test (n = 20)|
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Receptor for activated protein kinase C 1 negatively regulates gastric cancer tumorigenesis
To examine the effects of RACK1 on GC tumorigenesis, SGC-7901 cell line was selected for functional assays. The pCDNA3.1-RACK1 plasmid was transfected into the SGC-7901 cell to overexpress RACK1. On the other hand, we knocked down the intrinsic RACK1 expression using siRNA. The successful effects of overexpression and inhibition of RACK1 mRNA were demonstrated by real-time PCR [Figure 2]a. Cell viabilities were reflected by the growth rates measured by MTT assay. Importantly, we found that inhibition of RACK1 expression by its siRNA oligonucleotide dramatically promoted cell growth rate and vice versa [Figure 2]b. Our finding was consistent with the former report,  which strongly indicated a tumor suppressor role of RACK1 in GC cells.
|Figure 2: The effects of overexpressed and knockdown receptor for activated protein kinase C 1 on the cell viability in SGC-7901 cells in vitro. SGC-7901 cells were transfected with control vector, receptor for activated protein kinase C 1 plasmid, control small interfering RNA, and receptor for activated protein kinase C 1 small interfering RNA. Forty-eight hours after transfection, cells were harvested for expression test (a) and in the indicated time periods after transfection, cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays by measuring the absorption at 590 nm (b)|
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Receptor for activated protein kinase C 1 suppresses nuclear factor-kB signaling
To explore the effect of RACK1 on NF-kB signaling, we carried out the reporter assay to determine whether RACK1 overexpression could modulate the NF-kB signaling pathway. As shown in [Figure 3]a, overexpression of RACK1 led to decreased NF-kB signaling activity by approximately 2.5 folds (P < 0.01). Moreover, as shown in [Figure 3]b, after RACK1 was overexpressed, the mRNA expression of the NF-kB downstream transcription targets including A20, IkBα, and IL-8 appeared significantly downregulation, which further validating the observation from [Figure 3]a. Therefore, we suspected that the negative regulation of the pro-oncogenic NF-kB activity by RACK1 might contribute to its tumor suppressor role in GC cells.
|Figure 3: Receptor for activated protein kinase C 1 inhibits nuclear factor-ƒÈ B signaling. (a) Overexpression of receptor for activated protein kinase C 1 attenuated nuclear factor-ƒÈ B activity. SGC-7901 cells were transfected with nuclear factor-ƒÈ B-Luc, ƒÀ -galactosidase, and receptor for activated protein kinase C 1 plasmids. In 24 h, luciferase activity was determined, and relative nuclear factor-ƒÈB activity was calculated. ** P < 0.01. (b) Receptor for activated protein kinase C 1 repressed transcription of endogenous A20, IƒÈ Bƒ¿ , and interleukin-8 genes. SGC-7901 cells were transfected with receptor for activated protein kinase C 1 plasmids for 24 h, and then, the cells were harvested for real-time polymerase chain reaction|
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| » Discussion|| |
In this study, we examined the expression of RACK1 in human GC samples. Consistent with the former studies, , in our system, RACK1 was downregulated in GC tissues compared with the matched normal tissues. In addition, Deng et al. showed RACK1 was also decreased in a panel of GC cell lines when compared with the normal gastric cells.  According to their results, we selected SGC-7901 cells for further analysis in our system. Importantly, we characterized in our study that RACK1 could limit cell viability reflected by cell growth arrest. Moreover, to explain its growth inhibitory capacity in GC cells, we perform luciferase reporter assay as well as real-time PCR to reveal the impact of RACK1 overexpression on the NF-kB signaling activity. The results came out that RACK1 could inhibit the NF-kB signaling, which is the 1 st time indicating its modulation on this pathway.
NF-kB is a heterodimer transcription factor, which mainly contains p65 and p50 proteins, and could induce inflammatory cytokines and antiapoptotic proteins expression. Increasing evidence reported that NF-kB activation is associated with apoptosis resistance and carcinogenesis due to its fundamental roles on cellular dedifferentiation and proliferation in malignancies.  In our study, we pointed out that RACK1 could repress the NF-kB activity. This negative regulation could at least partially explain the role of RACK1 in GC. However, direct or intensive evidence is still lacking to explain this kind of regulation. In the following studies, substantial work should be done to dig out more about this regulation. Since RACK1 acts as a vital adaptor in various signaling pathways, is it possible that its interacting partners be involved in this regulation?
With the capacities of binding with a diversity of proteins, RACK1 was known to influence a wide variety of cellular processes. In our study, we just detect its impact on cell growth. Other biological processes, such as metastasis, apoptosis, and angiogenesis, should also be investigated in the following experiments to fully understand its role in GC tumorigenesis. Meanwhile, in vivo study is also essential to prove its crucial role in GC tumorigenesis.
Taken together, our study concluded that RACK1 was downregulated in human GC samples and acted as a potential tumor suppressor by inhibiting cell viabilities. Mechanically, we first demonstrated that the NF-kB signaling might contribute to its growth inhibitory effect in GC cells. Further study of RACK1 may provide novel promising therapeutic targets in GC treatment.
Financial support and sponsorship
This study is supported by Kaifeng Science and Technology Development Project (NO. 1403005).
Conflicts of interest
There are no conflicts of interest.
| » References|| |
Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64:9-29.
Hartgrink HH, Jansen EP, van Grieken NC, van de Velde CJ. Gastric cancer. Lancet 2009;374:477-90.
Wu CW, Hsiung CA, Lo SS, Hsieh MC, Chen JH, Li AF, et al.
Nodal dissection for patients with gastric cancer: A randomised controlled trial. Lancet Oncol 2006;7:309-15.
Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277-300.
Deng JY, Liang H. Clinical significance of lymph node metastasis in gastric cancer. World J Gastroenterol 2014;20:3967-75.
Dassen AE, Lemmens VE, van de Poll-Franse LV, Creemers GJ, Brenninkmeijer SJ, Lips DJ, et al.
Trends in incidence, treatment and survival of gastric adenocarcinoma between 1990 and 2007: A population-based study in the Netherlands. Eur J Cancer 2010;46:1101-10.
GASTRIC (Global Advanced/Adjuvant Stomach Tumor Research International Collaboration) Group, Oba K, Paoletti X, Bang YJ, Bleiberg H, Burzykowski T, et al.
Role of chemotherapy for advanced/recurrent gastric cancer: An individual-patient-data meta-analysis. Eur J Cancer 2013;49:1565-77.
Shimizu T, Marusawa H, Watanabe N, Chiba T. Molecular pathogenesis of Helicobacter pylori
-related gastric cancer. Gastroenterol Clin North Am 2015;44:625-38.
Xia Y, Shen S, Verma IM. NF-kB, an active player in human cancers. Cancer Immunol Res 2014;2:823-30.
Tkach KE, Oyler JE, Altan-Bonnet G. Cracking the NF-kB code. Sci Signal 2014;7:pe5.
Nagel D, Vincendeau M, Eitelhuber AC, Krappmann D. Mechanisms and consequences of constitutive NF-kB activation in B-cell lymphoid malignancies. Oncogene 2014;33:5655-65.
Sen R, Baltimore D. Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism. Cell 1986;47:921-8.
Karin M, Delhase M. The I kappa B kinase (IKK) and NF-kappa B: Key elements of proinflammatory signalling. Semin Immunol 2000;12:85-98.
Karin M, Cao Y, Greten FR, Li ZW. NF-kappaB in cancer: From innocent bystander to major culprit. Nat Rev Cancer 2002;2:301-10.
Lamb A, Chen LF. Role of the Helicobacter pylori
-induced inflammatory response in the development of gastric cancer. J Cell Biochem 2013;114:491-7.
Fan Y, Mao R, Yang J. NF-kB and STAT3 signaling pathways collaboratively link inflammation to cancer. Protein Cell 2013;4:176-85.
Hu F, Tao Z, Wang M, Li G, Zhang Y, Zhong H, et al.
RACK1 promoted the growth and migration of the cancer cells in the progression of esophageal squamous cell carcinoma. Tumour Biol 2013;34:3893-9.
Li JJ, Xie D. RACK1, a versatile hub in cancer. Oncogene 2015;34:1890-8.
Shen F, Yan C, Liu M, Feng Y, Chen Y. RACK1 promotes prostate cancer cell proliferation, invasion and metastasis. Mol Med Rep 2013;8:999-1004.
Wu J, Meng J, Du Y, Huang Y, Jin Y, Zhang J, et al.
RACK1 promotes the proliferation, migration and invasion capacity of mouse hepatocellular carcinoma cell line in vitro
probably by PI3K/Rac1 signaling pathway. Biomed Pharmacother 2013;67:313-9.
Chen L, Min L, Wang X, Zhao J, Chen H, Qin J, et al.
Loss of RACK1 promotes metastasis of gastric cancer by inducing a miR-302c/IL8 signaling loop. Cancer Res 2015;75:3832-41.
Deng YZ, Yao F, Li JJ, Mao ZF, Hu PT, Long LY, et al.
RACK1 suppresses gastric tumorigenesis by stabilizing the ß-catenin destruction complex. Gastroenterology 2012;142:812-23.e15.
Umezawa K. Inhibition of tumor growth by NF-kappaB inhibitors. Cancer Sci 2006;97:990-5.
[Figure 1], [Figure 2], [Figure 3]