Cis Platinum Induces Apoptosis and Supresses Invasion of Human Oral Tongue Cancer Cells (SP-C1) in vitro

Wisda Septiana Chandra Devi*, Ninik Nursanti*, Supriatno** *Gadjah Mada University Faculty of Dentistry
**Gadjah Mada University Faculty of Dentistry Academic Staff

Background ; Cis-platinum is a broad spectrum of neoplastic agent that is widely used to suppress the growth of human oral tongue cancer cells by induction of apoptosis. Apoptosis or programmed cell death has an important role in regulating physiological and pathological conditions. In the present study, the role of cis-platinum inducing apoptosis and inhibiting invasion or metastasis of human oral tongue cancer cells through the extrinsic and intrinsic pathway was analyzed. Methods ; The method used in this study was an experimental laboratory. To determine the proteolytic activity of caspase-3 (extrinsic pathway) and caspase-9 (intrinsic pathway), we used colorimetric test using a colorimetric assay kit (Ac-DEVD-pNA and AcLEHD-pNA). Boyden chamber was used to determine the ability of SP-C1 cell invasion after treated by cis-platinum. Results ; SP-C1 treated by 1 and 2 g/ml cis platinum showed increased proteolytic activity of caspase-3 and caspase-9 significantly. Concentrations of cis-platinum 1 and 2 g / ml also showed increased inhibition of SP-C1 cell invasion. Conclusion ; Cis - platin had a potential effect to induce an apoptosis activity and to inhibit human oral tongue cancer cells (SP-C1) invasion. Induction of apoptosis was occured through extrinsic pathway (receptor) and intrinsic pathway (mitochondria). Keywords ; cis-platinum, SP-C1, caspase-3 and-9, apoptosis, invasion

INTRODUCTION Apoptosis or programmed cell death has a regulating function of physiological, pathologic, and cytotoxicity condition of chemotherapy 1. There are two pathways of apoptosis mechanism, through receptors and mitochondria 2. Receptor mechanism begins with the stimulation of death receptors by Tumor Necrotizing Factor (TNF) receptor family that bind and attract Fas-associated Death Domain (FADD) and caspase-8 molecules 3,4. Activation of caspase-8 triggers the execution of apoptosis of the cell by effector caspase ( including caspase 3,6,7) cleavage 5. Mitochondrial mechanism initiate by the release of cytochrom-c from mitochondria. Once cytochrome-c is released it binds with Apoptotic Protease Activating Factor - 1 (Apaf1) and ATP, which then bind to pro-caspase-9 through caspase recruitment domain (CARD) 6 ,to create a protein complex known as an apoptosome 7. The apoptosome cleaves the procaspase-9 to its active form of caspase-9, which in turn activates the effector caspase-3 8.

Effector caspase activity playing an important role in regulating the induction of apoptosis in various types of cancer cells, including human oral tongue cancer cells. Oral tongue cancer is one of the worlds main health problems, as indicated by high incidence rates in some countries in the world. Oral tongue cancer most occured in developing countries 9, and characterized as an invasive and metastasis to regional cervical lymph nodes and often cause recurrence despite radical surgery was performed. This occurs because the micro-invasion of cells and metastasis of the primary cancer site 10. So far, treatment for oral tongue cancer cells still done conventionally, such as chemotherapy, radiotherapy, immunotherapy, surgery, and combination therapy which were not showing a significant increase in patients life expectancy. And also, during the last 10 years, the prognosis of oral tongue cancer cells hasnt been changed.11 . This fact show that it requires further studies of adequate, efficient and safe chemotherapeutic method to cure oral tongue cancer cells, one of them is using cis-platinum. Cis-platinum is a platinum derivative that functions as a bifunctional alkyl in inorganic compound which is widely used for various types of tumors treatment. Cisplatinum treatment had a significant effectiveness in patients with head and neck cancer. Combination of cis - platinum and 5-FU in 120 hours infusion showed high effectiveness for head and neck cancer treatment. A clinical study on phase II showed a mean range of treatment response was 20-70% and mean of complete response was ranged between 0% to 27%. 12 The aim of this study was to analyzed cis-platinum role on induced apoptosis and supressed invasion of human oral tongue cancer cells (SP-C1) through intrinsic (caspase-3) and extrinsic (mitochondria) pathway in vitro.

METHODS Cancer cells and cell culture Cancer cells used in this study were human oral tongue squamous cell carcinoma cell lines (Supris clone 1 / SP-C1) cloned by Supriatno, DD.S, MDSc., Ph. D. Human oral tongue cancer cell (SP-C1) was cultured from the tongue squamous cell carcinoma medium differentiation, without invasion to muscle tissues. SP-C1 cancer cells cultured in Petri dish (Falcon,NJ,USA) with Dulbeccos modified Eagle medium (DMEM, Sigma-Aldrich, MO,USA) medium infused by fetal calf serum 10% (Moregate Bio Tech, Australia) , streptomycin 100 g/ml and penicillin 100 unit/ml (invitrogen, CA, USA). Cancer cells then incubated at 95% humidity, 37 C temperature and 5% CO2. 50 mg of cis-platinum was obtained from Sigma-Aldrich Co., Tokyo, Japan. Apoptosis Detection To detected apoptosis, colorimetric test was done. Caspase 3 and caspase 9 proteolytic activity was determined by colorimetric assay kit (Ac-DEVD-pNA and Ac-

LEHD-pNA colorimetric assay kit, Biovision research product, CA, USA) as informed in factory protocol. Cell extract at the same amount was prepared from SP-C1 cells treated with cis platinum concentration 1 and 2 g/ml for 48 hours. Cell incubated with substrate (DVEDpNA caspase 3 or LEHD-pNA caspase 9) for 2 hours at temperature of 370C.14 Absorbent was determined by wave length 405nm using microplate reader (Bio-Rad). Invasion Cell Test SP-C1 cells treated by cis-platinum concentration 1 and 2 g/ml was starvated with cell amount 5 x 105 cells/dish for 24 hours. Polyvinyl pyrrolidone membrane (PVP: Nucleopore Co., CA, USA) with pore size 8 m incubated in 0,1 mg/ml gelatin (Merck, KgaA, Germany) in 30 minutes. Lower part of Boyden chamber filled with 25 l DMEM 10% FCS and coated by PVP membrane. Upper part of Boyden chamber filled with 50 l suspension of SP-C1 cells treated by cis-platinum concentration 1 and 2 g/ml. After 24 hours incubation, membrane was stained with Giemsa solution (Ted Pella Inc, USA). Membrane placed on object glass and observed by the light-microscopes under 100-times magnification. Measurement was counted from 12 different viewfields in each sample.15 Statistical Analysis Statistical analysis was done with Stat Work for Macintosh program (Cricket Software, Philadelphia, PA, USA). Data were analyzed with One-way ANOVA , followed by Turkeyss test (t-test) with level of significance 95% (p<0,05)

RESULTS Caspase-3 and Caspase-9 Activity SP-C1 cancer cells treated with cis platinum concentration 1 and 2 g/ml showed increase caspase-3 and caspase-9 proteolytic activity , compared with controls. As shown in Figure 1A, caspase-3 proteolytic activity in cis-platinum concentrations 1 g/ml has apoptotic ability of 1.65 fold, while the concentration of 2 g/ml was 1.90 fold compared with controls. Similar results also shown by caspase-9 proteolytic activity. SP-C1 cells treated with cis platinum concentration 1 g/ml has apoptotic ability of 1.40 fold, while the concentration of 2 g/ml was 1.77 fold compared with controls (Figure 1B). Statistical analysis showed a significant difference of caspase-3 and caspase-9 proteolytic activity in SPC1 cells treated with cis platinum concentration 1 and 2 g/ml. (P < 0,0045) SP-C1 cell invasion Cell invasion or cell migration is an important test to show the presence of invasion and / or metastasis of cancer cells15. SP-C1 cancer cells treated with cis platinum concentration 1 and 2 g/ml showed a very significant effect to supressed the ability of SPC1

cellular invasion,compared with controls (Figure 2). Statistical analysis results showed a significant difference of cellular invasion in SP-C1 cells treated with cis platinum concentration 2 g/ml. (P < 0,001)

DISCUSSION Cis-platinum derivative has several patent names, like carboplatin, cisplatin, vinorelbin, oxaliplatin, CDDP, and BBR3464. All these types of drugs have been widely used in clinics as chemotherapy or anti-neoplastic agent given by intravenous infusion 16. The level and severity of side effects that occur depends on the number and duration of cis-platinum administration. Increased duration, number and doses of cis-platinum, more severe side effects would occured, such as inflammation of the kidney and renal tubular damage. Cisplatinum is one of the most widely used chemotherapy in recent decades, because its effectiveness against some types of human cancer, include ovarian cancer17, head and neck18,endometriosis19, lung20, testis21, breast22, lymphoma23, and leukemia24. However, the mechanism of growth inhibition as well as other anticancer activity such as apoptosis, anti angiogenesis, and anti tumorigenesis still not known with certainty. The results show in Figure 1 indicates that the proteolytic activity of caspase-3 and 9 has increased very significantly in SP-C1 cancer cells treated with cis platinum concentration 2 g/ml. This data indicate that cis-platinum can induce apoptosis through both intrinsic and extrinsic pathway. Azuma et al25, reported that extrinsic pathway begins when cis-platinum enter cytoplasm membrane and binds to death receptors (FADD, TRADD, RIP, TRAF2, etc.) that stimulate caspase-8. Activation of caspase-8 would stimulate pro caspase3, -6, and -7 to activate caspase-3, -6 and -7 that caused apoptosis. Intrinsic pathway occurs after caspase-8 stimulate tBid activity in mitochondria.Afterwards, mitochondria release apoptosome in the form of cytochrom-c and Apaf-1 that binds to pro caspase-9 to activate caspase-9. Subsequently, caspase-9 will binds to effector caspase (caspase-3,-6,and-7), lead to apoptosis activity. SP-C1 cancer cell invasion after treated by cis-platinum, as shown in Figure 2 shows that there is a very significant increase in supression of cell invasion. These data indicate that cis-platinum significantly inhibits SP-C1 cancer cell invasion or migration though only administered in 24 hours. Similar results were reported by Ramer et al26, that cis-platinum effectively inhibit cell invasion through increase of regulation of tissue inhibitor of matrix metalloproteinase (TIMP-1) on human cervical carcinoma cells (HeLa). All results in this study showed that cis-platinum has the ability to inhibit the growth of human tongue cancer cells through induction of apoptotis activity and cell invasion. Conclusions of this study is cis-platinum is very effective in supress cell invasion and induce apoptosis of human oral tongue cancer cells. Apoptosis could occur through extrinsic (receptor) and intrinsic (mitochondria) pathway.

ACKNOWLEDGEMENT Our sincere thanks to Professor Mitsunobu Sato DDS.,Ph.D and Koji Harada DDS.,Ph.D who gave the facilities and infrastructure support infrastructure in order to complete the study. Authors also wish a gratitude to Supriatno, DD.S, MDSc., Ph. D who has given assistance to do this study.

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