Association between Expression of p70s6k with Radiotherapy Response in Cervical Cancer
I. Kurnia1*, B. Siregar2,Z. Alatas1, I. Ramli2, T. Kurjana3,A. Andrijono2, M.D.L. Tobing3, B.S. Hernowo3, T. Kisnanto1,D. Tetriana1,and S. Soetopo3
1Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebak Bulus Raya No.49, Pasar Jum’at, Jakarta 12440, Indonesia
2Cipto Mangun Kusumo Hospital,Jalan Diponegoro No.71, Jakarta10430,Indonesia
3Hasan Sadikin Hospital, Jalan Pasteur No.38, Bandung 40161, Indonesia
A R T I C L E I N F O A B S T R A C T AIJ use only:
Received date Revised date Accepted date Keywords:
Cervical Cancer Radiotherapy P70s6k
There aremany important prognostic factors in advanced stage cervical cancer primary treated with radiotherapy. Besides clinical factor, many biomarkers have been studied in relation with radiotherapy response. P70s6k is one of biomarker plays an important role in cell proliferation. Increased levels of p70s6kalso have associated with drug resistance of cancer. In the present study, wedetermined the association between the expressions of p70s6k before treatment with radiotherapy response in cervical cancer.Immunohistochemical staining ofp70s6k, MIB-1, p53 was conducted in microscopic slide from 21 cancer tissue biopsies before treatment with radiotherapy. After complete the treatment, early radiotherapy response was observed by pelvic control method. The resultsshow thatp70s6k is highly expressed(61.9%, 13/21) and low expressed (38.1%, 8/21) in the cancer cells andno significantdifference was foundonAgNOR mean, MIB-1 and p53 index in the different degrees of p70s6k expression (p≥0.05).High expression of p70s6k had association with good radiotherapy response compared tolower one (p=0.05). In conclusion, degree of p70s6k expression before treatment has association with radiotherapy response.
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INTRODUCTION
Cervical cancer was the most malignancy disease case found in Indonesia. Usually, patient come to the hospital was already at a local advanced stage. One of the treatment choices in local advanced staged form of this cancer is radiotherapy.
This treatment and sometime could be combined with chemotherapy or concurrent chemo- radiotherapy [1,2].
There are many important prognostic factors in advanced stage cervical cancer primarily treated with chemoradiotherapy are clinical pathologic
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factors, including stage and tumor histology [3].
Besides clinical pathologic factors, many biomarkers have been studied in relation to survival and/or response to chemoradiotherapy especially markers involved in tumorigenesis and tumor progression, such as genes associated with apoptosis, angiogenesis, and cell growth, that have been investigated extensively. At the moment, the focus of improving survival rates is mainly on targeted therapies in combination with standard chemoradiotherapy [4].
P70s6k is a serine/threonine protein kinase responsible for the phosphorylation and activation of 40S ribosomal subunit protein S6 [5,6]. It acts downstream of the mammalian target of rapamycin
(mTOR) in Akt pathway. The activation of p70s6k and its downstream target ribosomal protein S6 mediates nutrient and mitogen-stimulated translation, which is essential for cell growth and proliferation [7]. In some in vitro studies, it was foundthat activity of p70s6k related with prevention of apoptosis as one of death kind in cancer cell treatment with chemotherapy and in radiotherapy [8-11].
AgNOR, MIB-1 and p53 are biomarkers that related to cell proliferation. Nucleolar organizer regions (NORs) are chromosomal segments that carry ribosomal genes. Silver stained NORs are called AgNORs and when examined using light microscopy they appear as well-defined black dots exclusively located within the nucleoli [12]. MIB-1 is monoclonal antibody to detect Ki-67 antigen.
This antigen can bedetected in the nucleus of cells during the gap1 (G1), synthesis (S), and gap2 (G2) and mitosis (M) phases of the cell cycle [13]. Thus, it can be only detected in the nucleus of proliferating cells in all active phases of the cell- division cycle, but is absent in non-proliferating cells [14]. The p53 gene, located on chromosome 17, encodes a 53-kDa protein. Wild-type p53 protein is a transcription factor that, when activated, acts to arrest cells temporarily in the G1/G2 phase of the cell cycle allowing time for the repair of the damaged DNA, or to cause cells to proceed to apoptosis if the DNA damage is irrepairable [15,16].
In our previous publication there were correlation between apoptosis caspase-3 with better radiotherapy response, and contrary with high level of Akt expression that related with poor or bad radiotherapy response, but there were no association between some proliferation markers like AgNOR, MIB-1, p53, with radiotherapy response [17,18].
The aim of this study is to investigate association between p70s6k as biomarker with radiotherapy response in cervical cancer.
EXPERIMENTAL METHOD Patients
Twenty one consecutive patients, who achieved a complete treatment, from a whole series of 60 cases suffering from non-metastatic localized cervical carcinoma in local advance stage (IIB- IIIB), were prospectively conductedfrom July 2010 to March 2011. All patients were diagnosed and treated by definitive concurrent chemoradiotherapy in Cipto Mangunkusumo and Hasan Sadikin Hospitals. The treatment consists of external radiation and internal radiation. External Beam Radiotherapy (EBRT) with C-60 with total doses 50
Gy in 25 fractions. Internal radiation in 192Ir High Dose-Rate Intracavitary Brachytherapy (HDR-
ICBT) using a
Microselectron(NucletronInternational, Amsterdam, Netherlands) followedby EBRT in two fractions (850 cGy/fraction).Cisplatin was administered at dose of 40 mg/m2 on days 1, 8, 15, 22, and 29 that given concurrently with the EBRT in 2 hours or less before the treatment for corresponding day. Process of diagnosis and treatment followed the Standard Operational Procedure (SOP) as show in our previous publication [17].
Before the studies were carried out, the research proposal was approved by the Research and Ethical Committee from Faculty of Medicine, Universityof Indonesia and the patients received written informed consent.
Radiotherapy Response
Radiation response was evaluated by radiotherapist and as grouped by Hong Criteria as follow [19]:
1. NRT response (no gross residual tu- mor): complete or nearly complete regres- sion of pelvic tumor, non specific fibrosis, or granulation over the cervix (called as good response).
2. GT response (gross residual tumor):
gross tumor or palpable nodularity on cervix, and/or palpable in duration on the parametrium (called as bad response).
AgNOR Staining and Immunohistochemical AgNORstaining and Immunohisto-chemical of MIB-1, p53 and p70s6k were performed on microscopic slide from biopsy cancer tissue sample before treatment with radiotherapy. The detail analysis method of MIB-1 and p53 was conducted with procedures as in our previous publication [20,21]. P70s6k expression is observed in the nucleus and cell cytoplasm. The degree of its expression was estimated blindly of the clinical patient characteristics and outcome,with microscope magnification (10x40) semi-quantitatively. The scored was from negative (−) to slightly positive (+), moderately (++) and strongly positive (+++) [22]. The score from negative to slightly positive was grouped as low expression and moderate to strong positive was grouped as high expression
Statistical Analysis
Analysis of variance (ANOVA) Test was used to analysescomparison between expression of
p70s6k and AgNOR mean, MIB-1 index and p53 index. Fisher Exact Test was used toanalyze between expressions of p70s6k with radiotherapy response. All the test use with p≤0.05 as statistical significant. Minitab 17 was used to data analysis.
RESULTS AND DISCUSSION
Expression of p70s6k was detectable as brown colour in the nucleus and cytoplasm of cancer cell as shown in Figure 1a and b.
Itsexpression can be observed in all of 21microscopic slides from cancer tissue slide before radiotherapy. Itcan be grouped into high expressed (61.9%, 13/21) and low expressed (38.1%, 8/21).In Table 1, there was no statistical different of AgNOR mean, MIB-1 and p53 index in high and low expression of p70s6k before treatment. Different with AgNOR mean that tend lower in high expression of p70s6k, both of MIB-1 and p53 index is seem higher in high expression of p70s6k than lower one (Fig.2 a,b,c). After radiotherapy there was statistical different in proportion of complete and partial radiotherapy response in high and low expression of p70s6k (p=0.05) (Figure 3).
Tendency of low AgNOR meaninhigh expression of p70s6k is related with different activity site of p70s6k and AgNOR in part of cell cycle. AgNOR can be detected in G1, S and G2 in cell cycle and p70s6k expression is focused in G1only. The number of AgNOR start to be detected in G1, increased and accumulated in S and decreased again in G2 [23]. Another report also found that when compared in cancer tissue before treatment and after 9 Gy irradiated AgNOR mean is lower in after 9 Gy irradiated as considered in G2 phase and S phase beforeirradiation[21]. The similar data was also foundwhenre AgNOR observed in stimulated mitotic of hepatocyte cell [20]. As a mitogen-activated Ser/Thr protein kinase, p70s6k is required for G1 cell cycle progression and cell proliferation [8].
High expression of p70s6k related with cell proliferation. In active proliferation cell, amount of cell inmitotic (cycling cell) is higher than G0 phase.
Its clearlythat higher expression of p70s6k also show higher of MIB-1 index[24,25]. Higher MIB- 1 also mean that higher of growing fraction of cancer cell[21].
Even though without DNA sequencing analysis, almost expression of p53in cervical cancerwas in mutant than wild type.It was the main reason for this current data explaining higher p53 index in high expression of p70s6k than in lower one. Invitro investigation reported that wild type
p53 can decrease of p70s6k expression by mTOR pathway[26,27,28].
In this current result,high expression of p70s6k made the high proliferated cell and also prevents the process of apoptosis as one of way the death of cancer cell after receiving exposure ofioniczing radiation in radiotherapy. High portion of proliferating cell also made the cells to be more radiosensitive than radioresistance. A number of studies have focused on the involvement of p70S6k in influencing cellular responses to apoptotic stimuli. Treatment of Swiss 3T3 and RAT-1 cells with etoposide and staurosporine resulted in dephosphorylation and decreased activity of p70S6kin apoptosis preventing[8,9]. It has also been shown that cisplatin inhibited the phosphorylation of p70s6k in mouse myoblasts [10].
Rapamycin, an immune-suppressant blocked the activation of mTOR/p70s6k, thus compromising the cell's ability to progress through the G1 phase of the cell cycle [10,11]. On other in vitro study, using breast cancer cell,inactivated of p70s6k also let the process of cell death and should sensitize cancer cell to chemotherapeutic agents[29].
Cell in high proliferationwere highly radiosensitive and giving good radiotherapy response, in otherwise lower proliferation proportion will give the bad radiotherapy response.
Because in this result the observation of radiotherapy response as early response, we think that major of death of cancer cell was almostcausedby irradiation than cisplatin in concurrent therapy.
This present result seems have different with some reports explaining relation between expression of p70s6k with resistance of some cancer cells after treatment. Highexpression of p70s6k before treatment related with resistanceor poorresponse of cancer, especiallyin cancer treated with chemotherapy [30-32].
In the future, ifthis result will be able to be confirmed in bigger sample,we suggested the higher expressed p70s6k patients as one of consideration parameter receiving earlier treatment priority than the lower one. It also haspotential using anti p70s6k as target therapy in the form of adjuvant therapy after the patients completed the treatment of radiotherapy in cervical cancer.
CONCLUSION
Expression of p70s6k have important role in cell cancer proliferation to make the cancer tobe more radiosensitive or radioresistantand have potential to predict the radiotherapy response and
also as a consideration parameter in selection patientbefore treatment.
ACKNOWLEDGMENT
This research was supported by a Research Grant from Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Fiscal Year 2011. The authors thank Dr.
MukhSyaifudin, Head of Radiobiology Group, Division of Nuclear Medicine and Radiation Biology,Center for Technology of Radiation Safety and Metrology, National Nuclear EnergyAgency for reviewing and editing this manuscript.
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If you find difficulties in placing figures or table in the text above, please insert them in this page!
Fig 1. Expression of p70s6k in cytoplasm (a) and nucleus (b) in cervical cancer before treatment with radiotherapy (originalmagnification 10 x 40).
Table 1.AgNORmean, MIB-1 index and p53 index in different expression of p70s6k Biomarker Low Expression p70s6k High Expression p70s6k p value
AgNOR Mean 6.01±1,23 5,33±0,94 0.17ns
MIB-1 Index 0,39±0.10 0.47±0,13 0,16ns
P53 Index 0.34±0.13 0.46±0.16 0.11ns
ns:not significant (p>0.05)
Fig 2. AgNOR mean (a), MIB-1 indexs(b) and p53 indexs (c) in different expression of p70s6k in cervical cancer before treatment with radiotherapy.
Fig 3. Expression of p70s6k in different radiotherapy response.
