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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 10  |  Page : 616-621

Pyruvate kinase M2 inhibits the progression of bladder cancer by targeting MAKP pathway


Department of Urology Surgery, Chinese PLA General Hospital, Beijing 100853, China

Date of Web Publication24-Sep-2018

Correspondence Address:
Baofa Hong
Department of Urology Surgery, Chinese PLA General Hospital, No. 28, Fuxing Road, Haidian District, Beijing 100853
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.187302

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 > Abstract 


Purpose: Pyruvate kinase M2 (PKM2) was found overexpressed in tumor cells and played a significant role in tumor formation and growth. We sought to explore the correlation of PKM2 expression with cell proliferation, invasion, and apoptosis in bladder cancer cell line.
Materials and Methods: Real-time polymerase chain reaction and Western blot were performed to determine the expression level of PKM2 at mRNA and protein level. MTT and flow cytometry were respectively used to explore the proliferation, invasion, and apoptosis in bladder cancer cell line in vitro.
Results: The results suggested that suppression of PKM2 significantly decreased the proliferation rate, invasive cell number, and migration ability of bladder cancer cells compared with blank group. Moreover, proteins such as MMP2 and MMP9 as well as P38 expression were also affected by the PKM2 expression changes. These results proved that PKM2 could be involved in the progression of bladder cancer by mitogen-activated protein kinases signaling pathway.
Conclusion: The data presented in this study revealed that PKM2 up-regulation may promote the development and metastasis of bladder cancer through promoting cell proliferation, migration and invasion via MAPK signal pathway.

Keywords: Bladder cancer, cell invasion, cell proliferation, mitogen-activated protein kinases pathway, pyruvate kinase M2


How to cite this article:
Zhu Q, Hong B, Zhang L, Wang J. Pyruvate kinase M2 inhibits the progression of bladder cancer by targeting MAKP pathway. J Can Res Ther 2018;14, Suppl S3:616-21

How to cite this URL:
Zhu Q, Hong B, Zhang L, Wang J. Pyruvate kinase M2 inhibits the progression of bladder cancer by targeting MAKP pathway. J Can Res Ther [serial online] 2018 [cited 2019 Sep 16];14:616-21. Available from: http://www.cancerjournal.net/text.asp?2018/14/10/616/187302




 > Introduction Top


Bladder cancer is a kind of highly prevalent urogenital disease, ranking fifth in the most common cancers worldwide.[1] In addition, with high mortality, bladder cancer is regarded as the second most common malignancy of the urinary tract after prostate cancer.[2],[3] The knowledge of the molecular pathways associated with bladder cancer carcinogenesis is crucial to identify new diagnostic and prognostic biomarkers.

Pyruvate kinase M2 (PKM2) is a key enzyme in the process of glycolysis, which has been detected widely exists in the organization during the embryonic period, and with the development of embryo, it would gradually be replaced by other subtypes in some organizations. Increased conversion of glucose to lactate is a key feature of many cancer cells that promotes rapid growth, meantime, PKM2 expression is increased and promotes lactate production in cancer cells. PKM2 catalytic activity modulation could regulate the synthesis of DNA and lipids that are required for cell proliferation and of NADPH that is required for redox homeostasis at the same time. Besides, PKM2 functions as a protein kinase and as a transcriptional captivator. Given its multiway effects on cancer biology, PKM2 represents an attractive target for cancer therapy.[4],[5] PKM2 was found overexpressed in tumor cells and played a significant role in tumor formation and growth. As a key glycolytic enzyme, PKM2 can interact with many different kinds of biological molecules, playing a crucial role in the growth, survival, and metabolism reprogramming of cancer cells. Besides the location of cytoplasm as a glycolytic enzyme and the location of nucleus as a protein kinase, extracellular PKM2 is present in serum and feces of tumor patients.[6],[7] Researches have reported that PKM2 are highly expressed in breast cancer, lung cancer, colon cancer and many other tumors.[8],[9] However, the study on the connection between PKM2 and bladder cancer were rarely reported. Thus, the PKM2 expression and its biological impact on bladder cancer exploration may provide new ideas on bladder cancer diagnosis and treatment.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway was found allowing cells to interpret a wide range of external signals and respond appropriately by generating a plethora of different biological effects.[10]

In this study, we preliminarily investigate the expression of PKM2 in bladder cancer cells. Comprehensive experimental methods were sued to analyze the effects of PKM2 expression on the biological processes such as proliferation, invasion, and apoptosis. The aim of this study was to explore the expression level of PKM2 in RT112 cell line and to elucidate the possible molecular mechanism. This study may provide a new understanding of the regulated mechanism of bladder cancer in MAPK signaling pathway and identifies potential avenues for the therapeutic intervention for this disease.


 > Materials and Methods Top


Cell culture

The UBC cell line RT112 (G2, DSMZ ACC 418) was available for cell culture experiments. Cells were grown and maintained at 37°C 5% CO2 and 92% of humidity in RPMI-1640 (RT112) and Iscove's modified Dulbecco's medium (hTERT-BJ1) nutrient medium supplemented with 10% of FCS (all media were derived from GIBCO Invitrogen Corp., Carlsbad, California, USA).[11]

Plasmids and small interfering RNA transfection

The shRNA-expressing plasmids specifically targeting PKM2 and control small interfering RNA (siRNA) (no silencing) were synthesized by GenePharma Co (Shanghai, China). A PKM2 expression vector (pcDNA3.1-PKM2) was constructed by subcloning the full-length wild-type PKM2 coding sequence into pcDNA3.1(+) and confirmed by sequencing. The empty construct pcDNA3.1 was transfected as a control. Cell transfections were conducted using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's protocol. Stable PKM2 transfectants were generated under G418 (Gibco, Paisley, UK) selection as described before.[12],[13]

Cell proliferation

Cell concentration of RT112 in logarithmic growth phase was adjusted to 5 × 104/mL, cultured in 96-well plates with 200 μL per well. After culture for 24 h, 48 h, 72 h, and 96 h. Each group had four repeats. Add to each well 20 μL of fresh medium with 0.5 mg/mL MTT 4 h before termination and continue incubation for 4 h. Add 200 μL dimethyl sulfoxide to each well. We took values for each well using 492 nm optical density. The experiment was repeated for three times.[14]

Wound-healing assay

Cell motility was evaluated using the in vitro wound-healing assay. Cells in exponential growth phase were grown in 24-well plates until they reached confluence. Using a 20 mL plastic pipette tip, we scraped three horizontal lines across the entire diameter at the bottom of each well inducing the “wound.” Cell media were removed, and the cells were gently rinsed three times to remove unattached cells. The wound area was photographed at 24 h after scraping. To compare the cell motility of breast cancer cells, we measured the gap distance and determined the wound-closing rate. The cells were allowed to migrate into the wounded area for 24 h. At the indicated time points, the wound closure was photographed by a camera (Model DXM1200, Nikon, Japan) attached to an inverted microscope (Eclipse TE300, Nikon, Japan).[15]

Cell invasion assay

In vitro invasive assays were conducted using the Millicell Cell Culture Insert with 8 μM-pore polyvinylpyrrolidone-free polycarbonate membranes (Millipore, USA) covered with BD Matrigel™ Matrix (15 μg/filter). After growing to 90% to 100% confluent cultures, RT112 cells parental and derived cell lines (5 × 105 cells/mL) were trypsinized and resuspended in serum-free RPMI-1640 medium containing 1% bovine serum albumin. From this single-cell suspension, the cells were seeded at 1 × 105 (200 μL cell suspension) in the upper chamber of the matrigel invasion assay insert. The lower companion plate well contained RPMI-1640 plus 20% fetal bovine serum. After 12 h of incubation, the cells on the upper side of the membrane were removed by wiping with a cotton swab. The cells that migrated to the lower side of the membrane were fixed using the methanol and stained with crystal violet for 30 min and then dissolved with 33% of acetic acid. The number of cells was indirectly quantitated by measuring the absorbance at 570 nm.[15]

Real-time polymerase chain reaction

Total mRNA was isolated from cells and islets as previously described (Fransson et al., 2014). Complementary DNA (cDNA) was produced using reverse transcriptase (iScript™ cDNA Synthesis Kit; Bio-Rad Laboratories). The expression levels of mRNAs were measured by SYBR green-based quantitative real-time polymerase chain reaction (RT-PCR) (SYBR Green Master mix; Thermo Scientific, Waltham, MA, USA).[16]

Western blot analysis

Protein samples (the same concentration per lane) were separated on a 10–12% sodium dodecyl sulfate polyacrylamide gel and blotted onto polyvinylidene difluoride membranes, blocked in poly (butylene succinate-co-butylene terephthalate)s (PBST) (0.1% triton in 19 PBS), and probed with primary antibodies overnight at 4°C. The membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. The immunoreactive protein bands were developed by enhanced chemiluminescence. The immunoreactive bands were analyzed by a densitometer.[17]

Mechanism analysis

Adding p38 inhibitor SB203580 (20 μM) in si-PKM2 and overexpression PKM2 cells, detecting the change of cell proliferation, migration, and invasion ability.

Statistical analysis

All experiments were repeated three times. The results of multiple experiments are presented as the mean ± standard deviation. Statistical analyses were performed using SPSS 19.0 statistical software (IBM, Chicago, USA). The P values were calculated using a one-way analysis of variance. P <0.05 was considered to indicate a statistically significant result.


 > Results Top


Pyruvate kinase M2 was highly expressed in RT112 cell line

The RT-PCR was performed to detect the expression changes of PKM2 mRNA of RT112 cell lines. The results summarized in [Figure 1]a. The PKM2 mRNA level was higher in RT112 than in control. To test the consequence of the reduced PKM2 expression in RT112 and detect the interference efficiency, the PKM2 specific siRNA (si-PKM2) were transfected. As is shown in [Figure 1]b, PKM2 expression was significantly decreased by the si-PKM2 transfection while it was highly expressed by the overexpressed PKM2 transfection.
Figure 1: The expression level of pyruvate kinase M2. (a) The expression of pyruvate kinase M2 mRNA in RT112 cell lines was tested by real-time polymerase chain reaction. (b) Small interfering-pyruvate kinase M2 was transfected into RT112, polymerase chain reaction, and Western blot were constructed to test the expression level of pyruvate kinase M2 mRNA. *P < 0.05 and **P < 0.01 compared with the control group

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Pyruvate kinase M2 suppression decreased cell viability

To further connection of the expression of PKM2 on cell viability, the cell viability was tested after si-PKM2 transfection by MTT. As shown in [Figure 2], the cell viability was significantly decreased after almost 72 h culture. Meantime, the overexpression of PKM2 notably promotes the cell viability.
Figure 2: Supression of pyruvate kinase M2 regulated the cell viability of bladder cancer cells. The cell viability was tested after each 24 h

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Pyruvate kinase M2 suppression inhibited cell migration

The wound-healing assay was constructed to explore the relevance between PKM2 and cell migration. The number of migrated cells was decreased after si-PKM2 transfection [Figure 3]a. At the same time, we had carried out another comparative study. In this procedure, we transfected the p38 inhibitor SB203580 and tested the relative cell migration. As shown in [Figure 3]a and [Figure 3]b, the transfection of SB203580 showed an almost same status of cell migration with the si-PKM2 group. This implied p38 a most possible signaling pathway of PKM2 in the regulation of bladder cancer cell migration.
Figure 3: Connection between cell migration and pyruvate kinase M2. (a) Migration rate was measured after transfection. (b) Group SB203580 was transfected with inhibitor of p38; group small interfering-pyruvate kinase M2+ SB203580 was cells interfered by small interfering-pyruvate kinase M2 transfected with inhibitor of p38; group pyruvate kinase M2+ small interfering-pyruvate kinase M2+ SB203580 was cells interfered by small interfering-pyruvate kinase M2 and transfected with inhibitor of p38, following by overexpression of pyruvate kinase M2. **P < 0.01 and ***P < 0.001 compared with the control group

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Pyruvate kinase M2 suppression prevented cell invasion

Invasive assays in vitro were conducted to demonstrate the correlation of PKM2 expression with relative cell invasion. The invasive cell number significantly decreased after si-PKM2 transfection [Figure 4]a and [Figure 4]b, suggesting that the suppression of PKM2 suppression could significantly decrease the invasive ability. Interestingly, the SB203580 transfection group showed consistent with the si-PKM2 group of cell invasion.
Figure 4: Connection between cell invasion and pyruvate kinase M2. (a and b) The number of cells was indirectly quantitated by measuring the absorbance at 570 nm. Group SB203580 was transfected with inhibitor of p38; group small interfering-pyruvate kinase M2+ SB203580 was cells interfered by small interfering-pyruvate kinase M2 transfected with inhibitor of p38; group pyruvate kinase M2+ small interfering-pyruvate kinase M2+ SB203580 was cells interfered by small interfering-pyruvate kinase M2 and transfected with inhibitor of p38, following by overexpression of pyruvate kinase M2. **P < 0.01 and ***P < 0.001 compared to the control group. Each assay was done in triplicate and repeated at least thrice

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Pyruvate kinase M2 suppression decreased the cell migration-related protein expression

To investigate the effects of PKM2 expression on cells at the molecular level, the expressions of MMP2 and MMP9 were analyzed by Western blot assay. [Figure 5]a and [Figure 5]b shown MMP2 and MMP9 relative density separately, suggesting that PKM2 suppression could notably down-regulated the expression of MMP2 and MMP9. Thus, the expression of PKM2 changed the related protein expression. In addition, we further verified the possible pathway that PKM2 may be involved in. Hence, we researched the p38 and p-p38 expression level after suppression and overexpression of PKM2. Interestingly, the p38 was promoted after the knocking down of PKM2 while p-p38 shown opposite tendency. To further confirm the signaling pathway of PKM2, SB203580, inhibitor of p38 was transfected. [Figure 5]c and [Figure 5]d shows the MMP2, MMP9, and the p38 expression level after the transfection of SB203580 in each group. These results revealed that the influences of PKM2 expression on bladder cancer cells may be associated with the p38 signal.
Figure 5: Pyruvate kinase M2 expression connected with MMP2 and MMP9 expression. Pyruvate kinase M2 expression influence the p38 level. (a and b) MMP2, MMP9, and p38 expression were measured of groups of small interfering-pyruvate kinase M2 and small interfering-pyruvate kinase M2+ pyruvate kinase M2. (c and d) MMP2, MMP9, and p38 expression were measured of groups of small interfering-pyruvate kinase M2+ SB203580 and pyruvate kinase M2+ small interfering-pyruvate kinase M2+ SB203580. Each assay was done in triplicate and repeated at least thrice. Error bars indicate means ± standard deviation and *P < 0.05, **P < 0.01 and ***P < 0.001 compared with control group

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 > Discussion Top


Bladder cancer is the seventh most common cancer worldwide. Throughout the last decades, researchers have paid much attention on the treatment and pathology of the carcinogen including the risks of bladder cancer or the potential signaling pathway.[18],[19],[20] Recent evidence reveals that PKM2 is abnormally expressed in various kinds of diseases through involving in lots of biological processes such as cell proliferation, metabolism, and cancerization.[4],[21] In agreement with previous data,[22],[23] our results showed that PKM2 was highly expressed in the bladder cancer cells, indicating the correlation between PKM2 abnormal expression and bladder cancer progression.

Accumulating evidence shows that the activity of the PKM2 isoform is closely related to tumorigenesis. In this study, we preliminary discusses the role of PKM2 in bladder cancer. The expression of PKM2 is higher in bladder cancer cell line than in control cells, which is consistent with the investigation on the relationship between PKM2 expression and lung adenocarcinoma.[24] Then, we used small interfering RNA technology to investigate the effect of targeted PKM2 knockout on tumor growth at the cellular level. The results indicate that the PKM2 knockdown could significantly inhibit the cell proliferation, invasion, and migration ability in vitro. Further Western blotting analysis showed that knockdown of PKM2 inhibited the expression of MMP2 and MMP9. This finding is vital in elucidating the mechanisms by which PKM2 influences the growth and metastasis of bladder cancer at the cellular and molecular level.

p38 kinases are members of the MAPK with established contribution to a wide kind of signaling pathways and distinct biological processes.[25],[26],[27] The MAPK pathway has been proved to be related to many kinds of cancers, such as breast cancer.[28],[29] Our research focuses on the specific roles of p38 regarding cells proliferation, apoptosis in bladder cancer. The results demonstrate that p-p38 expression was obvious inhibited after knockdown of PKM2 while interference of p38 inhibitor shown a consistent expression level with the knockdown of PKM2. This suggests the role of PKM2 in bladder cancer cell line could be achieved by MAPK signal pathway of p38.

Taken together, the data presented in our study reveals that PKM2 was abnormally expressed in bladder cancer cell and its suppression may play certain prevent roles in the development and metastasis of bladder cancer by inhibiting the MMP protein and the p38 signal. This study may provide new potential therapeutic approach for the treatment of bladder cancer metastasis. However, further experimental studies for PKM2 expression in bladder cancer at the transcriptional level will be needed to verify our data.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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