Cancer Letters

Cancer Letters

Volume 276, Issue 2, 18 April 2009, Pages 189-195
Cancer Letters

RNA interference targeting the CD147 induces apoptosis of multi-drug resistant cancer cells related to XIAP depletion

https://doi.org/10.1016/j.canlet.2008.11.010Get rights and content

Abstract

CD147 (basigin, EMMPRIN) is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily. It is highly expressed on the surface of malignant tumor cells to promote their invasiveness and chemo-resistance. The present study aimed to reveal the anti-apoptotic effect of CD147 on the multi-drug resistant (MDR) phenotype of human oral squamous carcinoma cells (SCC) and its possible pathways. Data presented herein showed that MDR derivative SCC KB/V cell line expressed significantly higher CD147 and X-linked inhibitor of apoptosis (XIAP) than its sensitive counterpart KB cells by RT-PCR and Western blot analysis. Down-regulation of CD147 by transfection with CD147 siRNA resulted in decreased XIAP expression. Flow cytometric analysis and electron microscopic observation revealed differential cell apoptotic status related to CD147 expression. Additionally, chemo-sensitivity to 5-fluorouracil of KB/V was increased by CD147 silencing as measured by MTT colorimetric assay. These results suggest that inhibition of CD147 and subsequent XIAP depletion may have an anti-tumor effect through enhancing the susceptibility of cancer cells to apoptosis.

Introduction

CD147 is a member of the immunoglobulin superfamily enriched on the surface of many malignant tumor cells, which promotes multiple properties including growth, metastasis, and multi-drug resistance (MDR) of malignant cells [1], [2], [3], [4]. Recent work has revealed that CD147 plays a role in tumor MDR via different pathways. CD147 was shown to increase the expression of ATP-binding cassette (ABC) transporter families, such as MDR1 (for multi-drug resistance) [5], [6], [7]. CD147 affects drug resistance of MDR tumor cells by regulating the susceptibility to apoptosis in a hyaluronan dependent manner that was mediated via the PI3K and Erk1/2 pathways [8]. Other studies also revealed the apoptotic effects of CD147 on different tumor cells [9]. However, the precise mechanisms of CD147 on MDR tumor cells’ apoptosis remain to be understood.

Apoptosis resistance, which leads to survival of cancer cells, is the major hindrance in treatment of malignant tumor since most chemotherapeutic drugs are designed to induce apoptosis of cancer cells. Over-expression of inhibitor of apoptosis protein (IAP) which plays an important role in suppressing apoptosis via inhibiting caspase family [10], [11], [12] is the common mechanism of tumor cell apoptosis resistance. X-linked inhibitor of apoptosis (XIAP) is the most important member of IAP family and its enhanced expression is responsible for resistance to chemotherapy and radiotherapy, resulting in poor clinical outcome [13].

We previously demonstrated that the expression of CD147 was significantly lower in KB, a human oral squamous cell carcinoma (SCC) cell line, than that in KB/V, its MDR counterpart [14]. In the present study, we investigated the mechanism of the anti-apoptotic roles of CD147 in SCC drug resistance. RT-PCR and Western blot analysis were applied to determine the differential expression profiles of CD147 and XIAP as well as their internal regulation. Differential cell apoptotic status related to CD147 knock-down was demonstrated by flow cytometry (FCM) and electron microscope. The effect of CD147 on chemo-sensitivity of KB/V cells was confirmed by an anti-apoptotic agent 5-fluorouracil (5-Fu). Present results provide new understanding that CD147 plays a crucial role in MDR by regulating tumor cell apoptosis.

Section snippets

Cell culture

The human oral SCC cell line KB and its MDR derivative KB/V (KG-005, KG-127, KeyGen Biotech, Nanjing, China) were routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin-G, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. To maintain the MDR phenotype in the KB/V cell line, the medium was supplemented with 200 ng/mL vinblastine. Prior to start the experiments, KB/V cells were incubated for 10 days in drug-free medium.

Semiquantitative reverse transcription-PCR of multi-drug resistance-related proteins

Total

Significantly higher expression of CD147 and XIAP in KB/V cells

Total RNA from KB and KB/V were extracted and RT-PCR products were electrophoresed on 1.2% agarose gels. As shown in Fig. 1A, KB/V cells expressed equal levels of MDR1, MRP1, and LRP whereas significantly higher levels of CD147 and XIAP as compared with KB cells. Quantitative estimation showed that the expression of CD147 and XIAP were 2.4-fold and 3.3-fold higher in KB/V cells, respectively, than that of sensitive KB cells (Fig. 1B, p < 0.05).

Establishment of CD147 knock-down KB/V cells

Total RNA and protein from wild-type or transfected

Discussion

Resistance to multiple anticancer drugs is a major problem in treating malignant tumors. Most chemotherapeutic drugs exert their anticancer effects by inducing apoptosis [13]. CD147 was shown to up-regulate the expression of classic MDR related transporter, MDR1, and affect apoptotic pathways in cancer cells that enhance drug sensitivity. In cancer cells, CD147 regulates chemo-sensitivity to some chemotherapeutic drugs such as paclitaxel and curcumin [15], [16]. Thus, regulation of CD147

Conflicts of interest statement

None declared.

Acknowledgements

The study was supported by National Natural Science Foundation of China; Program for New Century Excellent Talents in University (NCET-05-0685); The Hunan Science Fund for Distinguished Young Scholars (06JJ-1005); Program for Creative Research for Ph.D. Candidate in University. We thank Ms. Tong Shen (St. John’s University, New York) for editing the manuscript.

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