Rab18 overexpression promotes proliferation and chemoresistance through regulation of mitochondrial function in human gastric cancer

Figures & data

Table 1 Distribution of Rab18 status in gastric cancer according to clinicopathological characteristics

Characteristics	Number of patients	Rab18 low expression	Rab18 high expression	P-value
Age				0.6047
<60	45	23	22
≥60	46	26	20
Gender				0.2573
Male	71	36	35
Female	20	13	7
Differentiation				0.4164
Poor	41	24	17
Well–moderate	50	25	25
Tumor invasion (T)				0.7348
T1+ T2	32	18	14
T3+ T4	59	31	28
Lymph node metastasis				0.0221
Absent	33	23	10
Present	58	26	32
TNM stage				0.0020
I	16	10	6
II	25	20	5
III	50	19	31

Figure 1 Expression of Rab18 in gastric cancers.

Notes: (A) Negative Rab18 staining in normal gastric tissues. (B) Weak cytoplasmic Rab18 staining in a sample of papillary gastric carcinoma. (C) Positive Rab18 staining in a sample of mucinous adenocarcinoma. (D) Positive Rab18 expression in a sample of tubular adenocarcinoma. (E) Positive staining in a sample of signet-ring cell carcinoma. (F) Rab18 staining in a sample of neuroendocrine carcinoma. (G) High Rab18 expression correlated with poor patient survival (log-rank test, P=0.0008).

Figure 2 Expression of Rab18 in gastric cancer cell lines.

Notes: (A) Western blot analysis of Rab18 protein in 10 samples of gastric cancer tissues with corresponding normal tissues. (B) Western blot analysis of Rab18 expression in gastric cancer cell lines (SNU-1, AGS, HGC-27, NCI-N87, MGC-803) and normal GES-1 cell line. (C) Plasmid transfection significantly upregulated Rab18 protein and mRNA levels in SNU-1 cells, and siRNA treatment downregulated Rab18 protein and mRNA expression in AGS cells.

Figure 3 Rab18 regulates proliferation, colony formation, and cell cycle.

Notes: (A) MTT assay showed that Rab18 overexpression promoted proliferation in SNU-1 cell line while Rab18 depletion inhibited proliferation in AGS cell line. (B) Colony formation assay demonstrated that Rab18 overexpression upregulated colony number in SNU-1 cell line while Rab18 depletion downregulated colony number in AGS cell line. (C) Cell cycle analysis demonstrated that Rab18 overexpression upregulated S phase percentage and downregulated G1 phase percentage in SNU-1 cells while Rab18 depletion upregulated G1 phase percentage and downregulated S phase percentage in AGS cell line. *P<0.05.

Figure 4 Rab18 regulates invasion, migration, and adhesion.

Notes: (A) Matrigel invasion assay demonstrated that Rab18 overexpression upregulated invading cell number in SNU-1 cells while Rab18 depletion downregulated invading cell number in AGS cells. (B) Wound healing assay demonstrated that Rab18 overexpression facilitated cell migration in SNU-1 cells while Rab18 depletion inhibited AGS cell migration. (C) Cell adhesion assay demonstrated that Rab18 overexpression downregulated cell adhesion in SNU-1 cells while Rab18 depletion increased the number of adherent AGS cells. *P<0.05.

Figure 5 Rab18 regulates CDDP-induced apoptosis in gastric cancer cells.

Notes: (A) MTT assay showed that Rab18 overexpression upregulated cell viability in SNU-1 cells after 24 and 48 hours of CDDP treatment while Rab18 depletion downregulated cell viability in AGS cells. (B) Annexin V/PI analysis demonstrated that Rab18 overexpression inhibited CDDP-induced apoptosis in SNU-1 cells while Rab18 depletion upregulated CDDP-induced apoptosis in AGS cells. The change of apoptosis rate was not significant in non-treated cells. *P<0.05.
Abbreviations: CDDP, cisplatin; PI, propidium iodide.

Figure 6 Rab18 regulates MMP and ROS production.

Notes: (A) JC-1 staining showed that Rab18 overexpression upregulated MMP with increased JC-1 red/green ratio in SNU-1 cells treated with CDDP, while Rab18 depletion downregulated MMP with decreased JC-1 red/green ratio in AGS cells treated with CDDP. (B) CellROX Deep Red staining showed that Rab18 overexpression reduced CDDP-induced ROS accumulation in SNU-1 cells while Rab18 depletion upregulated CDDP-induced ROS accumulation in AGS cells.
Abbreviations: CDDP, cisplatin; FITC, fluorescein isothiocyanate; MMP, mitochondrial membrane potential.

Figure 7 Rab18 regulates p-Rb, survivin, cytochrome c, cleaved PARP, and cleaved caspase 3.

Notes: Western blot showed that Rab18 overexpression upregulated the levels of p-Rb and survivin in SNU-1 cells while Rab18 depletion downregulated p-Rb and survivin levels in AGS cells. Rab18 overexpression downregulated cytochrome c, cleaved caspase 3, and cleaved PARP in CDDP-treated SNU-1 cells, while Rab18 depletion upregulated cytochrome c, cleaved caspase 3, and cleaved PARP in CDDP-treated AGS cells.
Abbreviation: CDDP, cisplatin.

Figure 8 Rab18 promotes cancer cell growth in vivo.

Notes: SNU-1 and AGS cells were stably transfected with Rab18 plasmid and Rab18 shRNA, respectively. Cells were injected into nude mice. CDDP was administered at doses of 5 mg/kg every 5 days; three injections of CDDP were given in total. Representative images of tumors and growth curves are shown.
Abbreviation: CDDP, cisplatin.

Figure S1 Validation of Rab18 antibody for Western blot and immunofluorescence.

Notes: (A) Western blot bands for Rab18 in cancer/normal tissues and AGS/AGS-siRab18 protein were of the same molecular weight. (B) Expression of Aurora B and Aurora B (phospho S227) in cells with Rab18 overexpression or knockdown. (C) Immunofluorescence of Rab18 in AGS and HGC-27 cells with relatively higher Rab18 expression. Cytoplasmic localization was observed in both cell lines. Localization of Rab18 staining could be observed around vesicles in AGS cells.

Figure S2 Immunohistochemistry for in vivo tumors formed by stably transfected cells.

Note: H&E staining, TUNEL assay, and immunostaining for Rab18, p-Rb, and survivin in tumor tissues formed by stably transfected SNU-1 and AGS cells.