Overexpression of GATA5 Stimulates Paclitaxel to Inhibit
Malignant Behaviors of Hepatocellular Carcinoma Cells
These authors contributed equally to this work.
Explore the effect of GATA5 expression on Paclitaxel inhibiting growth of hepatocellular carcinoma (HCC) cells.
Materials and Methods
In the experimental study, HCC cell lines (HLE, Bel7402 and PLC/PRF/5) were treated with different concentrations of Paclitaxel (5-20 mg/ml) for 24 hours. HLE cells were transfected with GATA5-siRNA vector, while Bel7402 and PLC/PRF/5 cells were transfected with overexpressed GATA5 vector for 24 hours, followed by treatment of the cells with Paclitaxel (10 mg/ml) for 24 hours and subsequently 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay to detect growth of HCC cells. Soft agar cultured was used to analyze formation of colony. Apoptosis of HCC cells were detected by Flow cytometer. Migration of HCC cells was observed by trawell assays. Western blotting and laser confocal microscopy were utilized to detect expression and location of the proteins.
Inhibiting expression of GATA5 reduced sensitivity of HLE cells to Paclitaxel, while overexpression of GATA5 increased sensitivity of Bel7402 cells and PLC/PRF/5 cells to Paclitaxel. Overexpression of GATA5 played a role in stimulating Paclitaxel to inhibit growth, colony formation and migration, as well as enhance apoptosis in HCC cells. Overexpression of GATA5 also promoted Paclitaxel to inhibit expression of reprogramming genes, such as Nanog, EpCAM, c-Myc and Sox2 in Bel7402 and PLC/PRF/5 cells. Inhibited expression of GATA5 led to enhancement of the expression of CD44 and CD133, in HLE cells. Overexpression of GATA5 was not only alone but also synergized with Paclitaxel to inhibit expression of CD44 and CD133 in Bel7402 or PLC/PRF/5 cells.
Overexpression of GATA5 played a role in enhancing Paclitaxel to inhibit the malignant behaviors of HCC cells. It was involved in suppressing expression of the reprogramming genes and stemness markers. Targeting GATA5 is an available strategy for applying paclitaxel to therapy of patients with HCC.
Paclitaxel is an effective chemotherapeutic drug that is widely applied in the treatment of a number of cancer types. It promotes cell death through apoptotic pathway (1, 2), while it causes drug resistance in the cancer cells (3). Hepatocellular carcinoma (HCC) is the fifth most frequent type of cancer and the rate of drug-resistance in HCC patients is high (3, 4). Surgery is considered as the best method for the treatment of liver cancer. However, many patients are diagnosed in the middle and late stages of disease and they loss chance of surgery. Thus, the mortality rate of liver cancer patients is higher than many other types of malignant tumor (3, 5). There is an imperative need to explore the mechanism of HCC cell resistance to drug therapy and to develop new strategy for treating drug-resistance of HCC patients.
GATA family regulates cell reprogramming to induce stem cell differentiation and normal
function of cells (6). This family includes GATA1-6, while GATA3 plays a key role in the
regulating breast cancer suppression (7). GATA5 also inhibits proliferation, invasion and
migration of cholangiocarcinoma cells (8). Hypermethylation of gene promoter suppresses
Previously, evidences indicated high expression of some reprogramming genes and stemness
markers in HCC cells (10-13). In this study, we investigated how GATA5 influenced
proliferation, apoptosis, migration and invasion of HCC cells after treatment with
Paclitaxel. The results displayed that overexpression of
Materials and Methods
In the experimental study, three human liver cancer cell lines (HLE, Bel7402 and PLC/PRF/5) were selected to test, the HCC cells were purchased from the Institution of Cellular Biology, Shanghai Academy of Life Science, China Academy of Science (Shanghai, China). These cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) at 37°C in a humidified atmosphere containing 5% CO2. The culture medium was replaced or the cells were passaged according to their growth state after 1-2 days. This study protocol was approved by the Ethical Committee of Hainan Medical College (code: 20170106).
Construction and transfection of the
GATA5 expression vector
The construct of stable expression vector CDH-
They were then ligated into the expression vector pCDH-CMV-MCS-EF1-coGFP (Systembio, USA)
by the HindIII and NotI restriction enzymes (Takara Bio Inc., China). The expression
vector was transfected into HCC cells by Lipofectamine 2000 (Invitrogen, USA). To obtain
the stable expression vector CDH-
Transfection of siRNA-
1.5×104 cells/ml of HLE, Bel7402, PLC/PRF/5, HLEsiRNA-
Analyses of the cell morphology, cell death and cellular nucleus
The HLE, Bel7402, PLC/PRF/5, HLE-siRNA-
Soft agar colony formation assay
Approximately 1000 cells were plated in the 6-wells plate and they were cultured in complete medium containing 20% FCS, mixing with 0.7% soft agar (1:1) to lay the upper layer. Then, the cells were incubated for 14 days at 37˚C. The colonies were photographed and counted using a Nikon inverted microscope (Nikon, Japan) (9).
Crystal violet staining observation of the colony formation
The cells were transferred into the fresh 6-wells plate Petri-dishes at a concentration of 800 cells/well, followed by growth selection using 400 mg/ml of G418 (Beijing Baiaolaibo Science and Technology Ltd., China). After 14 days of incubation, the cells were fixed with 75% ethanol for 30 minutes. They were subsequently stained with 0.2% crystal violet (Beijing Zhongshan Biotechnology Co., China) for colony visualization and counting (9).
The HLE, Bel7402 and PLC/PRF/5 cells were cultured in RPMI-1640 medium supplemented with
10% FCS at 37°C in a humidified atmosphere of 5% CO2. The cells were
transfected with siRNA-
Cells migration assays
The transwell method was used for observing the cells migration, and it was performed
according to the manufacturer’s protocol (Biofavor Biotech, China) and as described
previously (17, 18). The HLE, Bel7402 and PLC/PRF/5 cells (1.5×104) were
transfected with siRNA
To evaluate the effect of Paclitaxel on migration-related proteins, reprogramming genes
and stemness markers, the cells were transfected with siRNA-
Detection of proteins expression by laser confocal microscopy
Expression of the stemness markers (CD44 and CD133) was observed by laser confocal
microscopy after drug screening. HLE, Bel7402 and PLC/PRF/5 cells were transfected with
The data are presented as the mean ± SD. Statistical analysis was performed using Student’s t test (for two experimental groups) and F-test (SPSS 11.5 software for Windows, SPSS Inc., USA). The statistical significance was set at P<0.05.
GATA5 stimulated Paclitaxel to inhibit the growth of hepatocellular carcinoma cells
To investigate the influence of GATA5 on Paclitaxel regulating growth of HCC cells, we
first conducted a MTT assay to analyze the influence of different concentrations of
Paclitaxel (5-20 μg/ml) on proliferation of HCC cells. When the optimal concentration of
paclitaxel was determined as >10 μg/ml, growth of these HCC cells was significantly
inhibited (Fig .1A,). Then, we used Western blot to test GATA5 expression in the HCC cells.
Result showed that the HLE cells had high expression of endogenous GATA5, but the
endogenous expression of GATA5 in the Bel7402 and PLC/PRF/5 cells was low (Fig .1B,). Thus,
we silenced GATA5 expression in the HLE cells by transfecting the cells with
GATA5 enhanced Paclitaxel to promote apoptosis of hepatocellular carcinoma cells
In this study, we also investigated whether GATA5 was able to enhance Paclitaxel to
induce apoptosis of HCC cells by microscopy observations, trypan blue exclude staining,
DAPI staining and a flow cytometry analysis. The HCC cells were treated with Paclitaxel
for 48 hours followed by transfection with siRNA
GATA5 enhanced the effect of Paclitaxel on inhibiting migration and invasion of HCC cells
In this study, we also investigated whether GATA5 synergizes with Paclitaxel to inhibit
HCC migration by a transwell analysis. The microscopic observations showed that after
We also assessed the influence of GATA5 on the expression of the metastasis-related
factors, MMP2 and MMP9. In the present study, Western blot results indicated that after
silencing expression of
GATA5 increased the effect of Paclitaxel on inhibiting colony formation of hepatocellular carcinoma cells
In addition, we investigated whether GATA5 was able to enhance the effect of Paclitaxel
on inhibiting colony formation of the HCC cells. The crystal violet staining and
microscopy (×100) observations showed that HLE cells treated with Paclitaxel and
transfected with siRNA-scramble (Paclitaxel+siRNA-scramble group) inhibited colony
GATA5 increased the effect of Paclitaxel on inhibiting expression of the reprogramming genes
To investigate how GATA5 mechanistically stimulated Paclitaxel to suppress malignant
behaviors of HCC cells, we analyzed expression of the cancer stem cell reprogramming
genes, Nanog, EpCAM, c-Myc and Sox2 in the cells by Western blotting. The results
indicated that after silencing expression of GATA5 by transfecting the HLE cells with
GATA5 promoted Paclitaxel to inhibit expression of stemness markers, CD44 and CD133 in hepatocellular carcinoma cells
The stemness markers CD44 and CD133 play a key role in maintaining the malignancy of
HCC. Thus, in this study, we investigated whether GATA5 was able to play a role in
stimulating Paclitaxel to suppress expression of CD44 and CD133 in HCC cells. Western blot
was performed to assay expression of these proteins and laser confocal microscope
observation was applied to detect expression and location of these markers in the HCC
cells. The results indicated that silencing expression of
Paclitaxel is now widely used as a chemotherapeutic drug for treatment of many types of
cancer. It blocks the M/G2 cell cycle and stimulates caspase signal transduction to promote
apoptosis in cancer cells (22, 23). Due to the inherent or late acquired drug resistance of
liver cancer cells, sensitivity of HCC cells to Paclitaxel is reduced, limiting application
of Paclitaxel in the treatment of liver cancer (24, 25). Drug resistance in the liver cancer
cells is a crucial problem in clinical treatment. Our study showed that the endogenous
expression of GATA5 was higher in the HLE cells than in Bel7402 and PLC/PRF/5 cells. Thus,
we silenced expression of
To further demonstrate whether GATA5 synergized with Paclitaxel to suppress malignant behaviors of HCC cells, we performed HCC cellular colony formation, migration and invasion assays to assess the influence of GATA5 in the HCC cells accompanied by the treatment with Paclitaxel. Colony formation assay indicated that GATA5 synergizes with Paclitaxel to significantly inhibit cellular colony formation in the HCC cells. The cell migration and invasion assay indicated that GATA5 synergized with Paclitaxel to significantly reduce pore migratory capacity of the HCC cells and overexpression of GATA5 enhanced Paclitaxel to inhibit expression of the migrationrelated factors, MMP2 and MMP9. These results further demonstrated that GATA5 promoted Paclitaxel to induce apoptosis of HCC cells. Enhancing expression of GATA5 was able to synergize with Paclitaxel to inhibit HCC cells migration and invasion. GATA5 increased the sensitivity of HCC cells to Paclitaxel which maybe involved in suppressing expression of MMP2 and MMP9.
Cancer stem cells play pivotal role in malignant cells transformation (26, 27).
Reprogramming genes, such as
This is the first report indicating that GATA5 plays a role in promoting Paclitaxel to inhibit the growth, migration, invasion and colony formation of HCC cells, in addition to stimulating apoptosis in these cells. All together, we revealed that in terms of molecular mechanism, GATA5 synergizes with Paclitaxel to inhibit the malignant behaviors of HCC cells which maybe involved in suppressing expression of reprogramming genes and stemness markers. These findings suggest that enhanced expression of GATA5 may be an available strategy for applying Paclitaxel to treat HCC patients.
This work was supported by the National Natural Science Foundation of China (Nos. 81660463, 81560450, 31560243). The Natural Science Foundation of Hainan Province (Nos. 2019CXTD406, 20168263 and 814293); Hainan Provincial Association for Science and Technology Program of Youth Science Talent and Academic Innovation (No.QCXM 201922); The Hainan Graduate Students Innovate Program (Hys2017-178, Hys2017-180, Hys2018-277, Hys2018-278, Hys2018-279). The authors declare that they have no competing interest.
H.F., B.L., Y.Z., JX., Y.Z.; Performed the experiments. K.L., M.L.; Analyzed the clinical data and discussed the results. B.L., M.L.; Drafted the manuscript. M.Z., M.L.; Designed the experiments and revised the results. All the authors contributed to the manuscript editing and approval.