1477IntroductionThe traditional way to diagnose tumors is by histopathologystains and analysis. The diagnosis of cancer relies primarilyon invasive tissue biopsy [1]. The conventionalhistopathology based on light microscopy, however, hasrecently been complemented with ultrastructure, immunohistochemistryand molecular diagnostics [2].Cancer of the uterine cervix is the most common form ofcancer in women developing countries as leading cause ofcancer-related deaths in women in the world as a whole[3]. Cervical cancer is stay as the first for cancer causeddeath in Indonesia and still be a problem of health [4].There are four human viruses that cause cancer in human.There are papilloma viruses (PV), epstein barr viruses(EBV), hepatitis B virus (HBV), and kaposi sarcoma herpesvirus (KSHV) [3].The E7 protein binds to the underphosphorelated form ofthe tumor suppressor protein pRb and displaces the E2Ftranscription factor that is normally bound by pRb[3,5,6].The latent membrane protein-1 (LMP-1) of EBV preventsapoptosis of B cells by up regulating the expression of bcl-2, and it activates growth promoting pathway that are normallytriggered by T cell-derivated signal [3,5,6].

etm IntroductionCervical cancer is the third most commonly diagnosed cancer and the fourth leading cause of female mortality worldwide, with >85% of the associated deaths occurring in developing countries (1). The American Cancer Society estimates ~12,360 new cases of invasive cervical cancer and ~4,020 mortalities in the United States for 2014 (2). The oneyear survival rate for females with cervical cancer is 87% and the fiveyear survival rate is 68%. When detected early, the fiveyear survival rate for patients with invasive cervical cancer is 91% (3). Cervical cancer develops slowly, taking 1015 years to develop into cancer from a precancerous condition called dysplasia. Although fully treatable in the early stages, once the cancer has metastasized, patient outcome is poor.Critical to tumor cell invasion are the processes of cell attachment, proteolytic degradation of the extracellular matrix (ECM) and migration through the disrupted matrix (4). Rath and Pauling (5) proposed that the use of nutrients, such as lysine and ascorbic acid, to target plasminmediated connective tissue degradation should be considered as a universal approach to control tumor growth and expansion. Binding to plasminogen active sites, lysine blocks plasminogen activation into plasmin by tissue plasminogen activator; thus, it modulates the plasmininduced matrix metalloproteinase (MMP) activation cascade (6). We have previously developed strategies to inhibit cancer growth and its spread using complex micronutrient supplementation with select natural compounds, such as lysine, proline, ascorbic acid and green tea extract (7). This nutrient mixture (NM) demonstrated pleiotropic synergistic anticancer activity in vivo and in vitro in several cancer cell lines through the inhibition of cancer cell growth, MMP secretion, invasion, metastasis and angiogenesis (7).In previous studies we found that NM significantly inhibited the proliferation of cervical cancer HeLa cells in vitro, the secretion of MMP2 and 9, urokinase plasminogen activator activity and Matrigel invasion, and enhanced tissue inhibitor of matrix metalloproteinases 2 activity (8,9). In the present study the in vivo effects of NM supplementation ontumor growth and cancer markers in cervical cancer HeLa cell tumor xenografts in female nude mice were investigated. The cancer cell markers studied by tumor immunohistochemistry (IHC) were as follows: Ki67 (proliferation marker); MMP2 and 9 (metastasis markers); vascular endothelial growth factor (VEGF) (angiogenesis marker); terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and B-cell lymphoma 2 (Bcl2) (apoptosis markers); cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) (inflammatory markers) and glutathione S-transferase (GST) (specific cancer marker).

ONCOTAGEN

Human papillomavirus (HPV) infection can lead to a variety of human cancers [1, 2]. It has been shown that most cervical cancers are caused by HPV infection [3, 4]. Two oncogenes encoded in the HPV genome, E6 and E7 play critical roles in cervical cancer development [5]. Expression of HPV E6 leads to degradation of p53, which is a critical tumor suppressor regulating cell growth and apoptosis. Furthermore, HPV E7 binds and deactivates another important tumor suppressor, retinoblastoma protein (Rb). In most cases, expression of both E6 and E7 are required for oncogenic transformation. In addition to deactivating tumor suppressors p53 and Rb, HPV infection leads to many cellular changes during cervical cancer development [6]. However, the impact of HPV infection on host microRNA expression has not been well characterized. MicroRNAs (miRNAs) are small non-coding RNA molecules (~23 nucleotides) that downregulate the expression of their gene targets [7]. Both computational and experimental studies indicate that thousands of human protein-coding genes are directly regulated by miRNAs. Thus, miRNAs play important regulatory roles in many physiological processes as well as a variety of disease states such as cancer [8]. Relevant to this study, altered miRNA expression profiles have been reported in cervical carcinomas as compared with normal cervix [9-15]. In addition, miRNA expression changes have been used as biomarkers for cervical cancer prognosis [16]. However, it is not clear whether these cervical cancer-related miRNA changes were directly caused by HPV infection, or simply indirect effects from cervical cancer progression in general. To specifically characterize miRNA expression changes that are related to HPV activity, we determined the expression status of HPV E6 and E7 transcripts in 101 cervical carcinomas. Transcriptional activity of HPV was then correlated to miRNA expression profiles to identify HPV-associated miRNA changes. In this way, we have identified miR- 9 as the most activated miRNA by HPV E6 in cervical cancer, and showed that both the transcriptional activity of HPV E6/E7 and miR-9 were prognostic markers for cervical cancer. Further target validation and functional cell biology analyses showed that HPV-induced miR-9 activation led to significantly increased cell motility by downregulating multiple gene targets that are involved in cell migration, which may contribute to the progression of cervical cancer.

PONE 25Cervical cancer ranks the third among the most commoncancers in women worldwide and is the most common type ofcancer in Eastern Africa, South-Central Asia and Melanesia [1].The two major types are squamous cell carcinoma (SCC),accounting for around 80% of the cases, and adenocarcinoma(ADCA), for which the incidence has been increasing [24].Persistent infection with high risk (HR) human papillomavirus(HPV) is a major risk factor for the development of cervical cancerand the two predominant types are HPV18 and HPV16, followedby HPV45, 31, 33, 58, 52, 35, 59, 56, 6, 51, 68, 39, 82, 73, 66 and70 in order of prevalence [5].HR-HPV encodes two early proteins, E6 and E7, that targettwo major cellular tumor suppressor pathways. E6 targets the p53tumor suppressor for degradation, resulting in loss of p53-dependent apoptosis and/or senescence [6,7]. E7 binds to thepRb tumor suppressor, thereby disrupting G1/S transition control[8]. Consequently, HR-HPV infection may lead to malignanttransformation and tumor development.Genomic alterations such as gene amplifications are importantfeatures of cervical carcinogenesis [9,10]. Copy number gain in thelong arm of chromosome 3 has been shown to be a biomarker ofprogression from carcinoma in situ to invasive cancer [11]. Theamplified chromosome 3q region contains several genes withrelevance to cancer, such as the phosphoinositide-3-kinasecatalytic alpha polypeptide gene (PIK3CA) at 3q26.32 [12], thetelomerase RNA component gene (TERC) at 3q26.2 [13,14], theretinol-binding protein 1 and 2 genes (RBP1-RBP2) at 3q23, theTP63 gene at 3q28 [15], and the sex-determining region Y-box 2gene (SOX2) at 3q26.33 [1618].Human WIG-1 (wild type p53-induced gene 1; also namedZMAT3) maps to 3q26.32 [19], which is in the critical 3q region.WIG-1 is a bona fide p53 target gene [20]. It encodes a 288 aminoacid nuclear zinc finger protein that binds to double-strandedRNA with high affinity [19,21]. WIG-1 is highly conserved fromamoeba to human [22,23] and the Wig-1 protein has been shownto bind to a U-rich element in the 39-UTR of TP53 mRNA whichis then stabilized. Thus, Wig-1 forms a positive feedback loop withp53 that enhances p53 expression [24,25]. Wig-1 can also bind toand stabilize the MYCN oncogene mRNA [26], and targets anumber of other mRNAs [27]. Moreover, Wig-1 has been shownto destabilize CDKN1A mRNA (encoding p21) through recruitmentof the RISC complex [28]. WIG-1 is amplified and/or overexpressedin human papillary thyroid carcinoma (www.ebi.ac.uk/gxa/), lung squamous cell carcinoma [29], cervical SCC and otherhuman tumors (www.cbioportal.org), suggesting an oncogenicfunction.In this study we have examined the WIG-1 gene in cervicalcarcinoma cell lines and Wig-1 expression in both cervicalcarcinoma cell lines and tumor samples. We investigated theexpression of Wig-1 in relation to different clinical parameterssuch as: histological tumor type, grade and stage, HPV status, ageat diagnosis, and survival. Our results indicate that high nuclearWig-1 expression is a marker for poor prognosis in cervicalcarcinoma.

PONE 417Cervical cancer is worldwide the second most common cancer in women with the majority ofsquamous cell carcinoma (SCC) [1] resulting in 454,000 cases and 200,000 deaths per year in2010. Frequent metastatic sites are the pelvic lymph nodes, para-aortic lymph nodes, lung,extra-pelvic nodes, liver, and bones [2]. Approximately 11,000 new cases and 3,870 deathsoccur for cervical carcinoma in the U.S. [3]. Stage and nodal metastasis are related to overallsurvival [4]. Chemotherapy drugs used for cervical cancer include: paclitaxel, carboplatin, cisplatinum,bleomycin, mitomycin-C, vincristine and irinotecan [5]. Retinoids and interferon, incombination with cytotoxic chemotherapy, have been shown to be effective [6]. However,there is no standard treatment for metastatic cervical cancer. Therefore, a patient-like mousemodel of cervical cancer could be very useful.Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mousemodel with the technique of surgical orthotopic implantation (SOI) [721]. Unlike subcutaneous-transplant patient-derived xenograft (PDX) models, PDOX models metastasize. Most importantly,the metastasis pattern correlates to the patient.Histologically intact human colon-cancer specimens derived surgically from patients wereimplanted by SOI to the colon or cecum of nude mice. Extensive growth on the colon in 13 of20 cases of implanted patient colon tumors was observed with subsequent regional, lymphnode,and liver metastasis, as well as general abdominal carcinomatosis [7].SOI of histologically intact pancreatic-cancer specimens to the nude-mouse pancreas, resultedin a metastatic pattern that resembles the clinical pattern including local tumor growth,extending to the stomach and duodenum, metastases to the liver and regional lymph nodes,and distant metastases to the adrenal gland, diaphragm, and mediastinal lymph nodes. A 100%take rate was demonstrated for 5 cases, of a total 17 mice transplanted, 15 supported tumorgrowth. Immunohistochemical analysis of the transplanted human pancreatic tumors showeda similar pattern of expression tumor-associated glycoprotein 72 and carcinoembryonic antigenin the transplanted tumors and the original surgical biopsy [8].Histologically-intact patient specimens of ovarian cancer were developed by SOI under thecapsule of the nude mouse ovary. The tumors grew locally with a subsequent patient-like metastaticpattern, including the parietal peritoneum, colon, omentum, and ascites [10].Histologically-intact patient breast tumor tissue was transplanted to the mammary fatpad of nude mice by SOI where the tumor tissue grew extensively and metastasized to thelung [11].A patient-like metastatic model of human lung cancer constructed was developed with SOIvia thoracotomy in immunodeficient mice [9]. Tumors were transplanted into the left lung inall these experimental animals. The left lung was used for tumor implantation for 2 reasons:(1) the loss of lung function is smaller in the left lung than in right-lung during surgery. Theleft-lung-operated animals survive the procedure better. (2) The left lung in mice has onelobe, enabling tumors to readily develop after implantation [9].When a poorly-differentiatedlarge-cell squamous-cell patient tumor 2268 was implanted to the left lung by SOI directlyfrom surgery, 5 out of 5 mice produced locally-grown tumors, in an average time of 61 days.Opposite-lung metastases occurred, as well as lymph-node metastases. The primary tumorsand metastases in the mice maintained their large-cell-squamous-cell morphology.Whensubcutaneously implanted tumors grew only locally in 2 of 4 animals and no metastases wereobserved [9].In a clinical correlative study of 20 cases of stomach cancer that grew in nude mice, 5 hadclinical liver metastases and all 5 cases resulted in liver metastases in the nude mice. Of the 20cases, 6 had clinical peritoneal involvement of their tumor, and of these, 5 resulted in peritonealmetastasis in the nude mice. There were statistically significant correlations for both liver metastasesand peritoneal involvement between patients and mice [12].In the present report, we describe the development of a PDOX model of HER-2-positivecervical cancer with a metastatic pattern similar to the patient donor.

Kanker serviks adalah kanker yang paling banyak diderita oleh wanita diseluruh negara dan merupakan salah satu penyebab utama kematian akibat kanker pada wanita di dunia secara keseluruhan. Kanker serviks juga merupakan kanker yang menyebabkan kematian utama pada wanita di Indonesia dan masih menjadi masalah kesehatan. (1477) Tingkat kelangsungan hidup satu tahun kedepan untuk wanita dengan kanker serviks adalah 87% dan tingkat kelangsungan hidup lima tahun kedepan adalah 68%. Bila terdeteksi dini, tingkat kelangsungan hidup lima tahun kedepan untuk pasien dengan kanker serviks invasif adalah 91% . Kanker serviks berkembang lambat, sekitar 10-15 tahun untuk berkembang menjadi kanker dari kondisi pra kanker (ETM)

Kanker serviks disebabkan oleh infeksi HPV. Dua onkogen dikodekan dalam genom HPV, E6 dan E7 memainkan peran penting dalam perkembangan kanker serviks. Ekspresi HPV E6 menyebabkan degradasi p53, yang merupakan penekan tumor yang mengatur pertumbuhan sel dan apoptosis. Selain itu, HPV E7 mengikat dan menonaktifkan penekan tumor lain yang penting seperti protein retinoblastoma (Rb). Dalam kebanyakan kasus, ekspresi kedua E6 dan E7 yang diperlukan untuk transformasi onkogenik. Selain menonaktifkan penekan tumor p53 dan Rb, infeksi HPV menyebabkan banyak perubahan sel selama perkembangan kanker serviks (ONCOTAGEN).

Dua jenis utama kanker serviks adalah karsinoma sel skuamosa (SCC) sekitar 80% dari kasus, dan adenokarsinoma (ADCA), yang angka kejadiannya telah meningkat. Infeksi persisten dengan resiko tinggi human papillomavirus (HPV) merupakan faktor risiko utama untuk pengembangan kanker serviks (Pone 25).

Kanker serviks di seluruh dunia mayoritas mengidap karsinoma sel skuamosa (SCC) sekitar 454.000 kasus dan 200.000 kematian per tahun di 2010. Metastasis tersering yaitu kelenjar getah bening panggul, kelenjar getah bening para-aorta, paru-paru, node ekstra-panggul, hati, dan tulang. Obat kemoterapi yang digunakan untuk kanker serviks meliputi paclitaxel, carboplatin, Cisplatinum, bleomycin, mitomycin-C, vincristine dan irinotecan. Retinoid dan interferon, di kombinasi dengan kemoterapi sitotoksik, telah terbukti efektif. Akan Tetapi,tidak ada pengobatan standar untuk kanker serviks metastatik (Pone 417)