Cellullar Plasticity and dedifferentiation: A Link Between Cancer Stem Cells, Hypoxia, Cell Injury, and inflammation

Cellular plasticity is the concept of bidirectional dynamics change cells differentiation degree which involved in the regeneration, repair and tissue turnover along the organism livespan. Cellular plasticity and dedifferentiation process are well documented in the discovery of iPCSs by introducing several transcriptional factors known as Yamanaka factor to terminally differentiated somatic cells and reverted into pluripotent state as the ESCs. iPSCs are able to exhibit ESCs differentiation potential which could produce ectodermic, mesodermic, and endodermic cell lineage. In tumour biology, the tumour plasticity also have a similar regulation and play an imporant role for maintaining tumour integrity and survival, particularly in maintaining CSCs population. Various study of cellular plasticity regulation has shown that various factors are involved, in example hypoxia, cell injury, and inflammation. Cells respond to hypoxia, cell injury, and inflammation by chemoattractant which attract repair cells to homing towards injured sites. The homing mechanism of stem cells involved EMT to facilitates migration of stem cells towards injured sites, thus leading to tissue regeneration. On the other hand, cancer metastasis also showed a connection with EMT process. EMT which showed a change in cell properties are linked to dedifferentiation and hypoxia response. Hypoxia condition has been known to preserve and both normal stem cells and CSCs stemness. HIF which protected from degradation in hypoxia condition interact with DNA by binding to HRE. HRE activation trigger transcription of numerous signalling protein which involved in stemness, cell proliferation and survival. Therefore it is concluded that cell injury, hypoxia, and inflammation could programmed cells to undergo dedifferentiation process and involved in EMT regulations. CSCs which resides insides heterogeneous tumour cells population are though to be dynamicly regulate itself in the quietscent and active state through dedifferentiation like the normal stem cells. Understanding how CSCs regulates its active an quietscent state dynamics could provide an important information for novel CSCs targeted therapy development

Dedifferentiation of MCF-7 Breast Cancer Continuous Cell Line, Development of Breast Cancer Stem Cells (BCSCs) Enriched Culture and Biomarker Analysis

BACKGROUND: Cancer stem cells (CSCs) eradication might serve as a robust approach for cancer eradication. MCF-7 as breast cancer continuous cell line is known to contain breast CSCs (BCSCs) for its capability to maintain its original tumor population. CSCs enriched culture is a fundamental tool for CSCs targeted therapy development. Effective and unsophisticated CSCs dedifferentiation protocol for producing CSCs enriched culture is needed.METHODS: MCF-7 cells were cultured initially in Dulbecco's Modified Eagle Medium (DMEM) low glucose medium then changed to DMEM:F12. Serum starvation was performed during each medium refreshment gradually with fetal bovine serum (FBS) concentration of 10%, 5%, 2.5% until reaching 1% FBS concentration. Stable MCF-7 culture was then adapted to serum free culture system, containing DMEM:F12, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and B27 supplement as dedifferentiation protocol for 18 days. Cluster of differentiation (CD)44 and CD24 double staining immunocytochemistry was performed to evaluate cell stemness.RESULTS: The population of cells expressing BCSCs markers (CD44+/CD24low) in non-adherent single cells subpopulation was significantly increased after the dedifferentiation procedure (70.39%) compared to control groups (0.71%) (p<0.05). In contrast, the expression of BCSCs marker in adherent single cells subpopulation and for both adherent and non-adherent mammosphere the BCSCs markers showed a stable expression.CONCLUSION: BCSCs enrichment of breast cancer cell cultures from MCF-7 breast cancer cell line can be performed. Breast cancer cell plasticity is observed during the dedifferentiation protocol. Development of dedifferentiation inducing protocols can serve as an important foundation for breast cancer therapy development through BCSCs elimination.KEYWORDS: breast neoplasms, cell line, dedifferentiation, immunohistochemistry, neoplastic stem cell

Circulating Plasma miRNA-21 as a Superior Biomarker Compared to CA 15-3: Assessment in Healthy Age Matched Subjects and Different Stage of Breast Cancer Patients

BACKGROUND: Carbohydrate antigens 15-3 (CA 15-3) is a conventional tumor marker in breast cancer, with low sensitivity and specificity. MicroRNA (miRNA)-21 showed its stability in circulation and could serve as powerful biomarker. The aim of this study was to evaluate miRNA-21 as breast cancer biomarker compared to CA 15-3 in Indonesian population.METHODS: Circulating plasma miRNA-21 expression was measured using qRT-PCR in 49 patients at various stages of breast cancer and 16 healthy controls. The relative expression value of miRNA-21 was calculated using 2-ΔΔCt. Meanwhile, CA 15-3 was quantified using electrochemiluminescence immunoassay (ECLIA) methods. The results of miRNA-21 and CA 15-3 plasma circulating expression were compared with controls at each stage and between stages of breast cancer.RESULTS: CA 15-3 median level in breast cancer group was 1.60 times higher compared to control group (p=0.019), 21.00 m/mL and 13.05 m/mL, respectively. Median miRNA-21 expression in breast cancer group was elevated 4.92 folds compared to control group (p=0.001), 4.43 and 0.90, respectively. There was no significant difference of CA 15-3 level between controls and all stages of breast cancer group. CA 15-3 cut-off value was 15.05 m/mL (p=0.016) with 59.2% sensitivity and 62.5% specificity. Meanwhile, there was a significant difference of miRNA-21 expression between controls and most stages of breast cancer group. Circulating miRNA-21 expression cut-off value was 2.07 (p=0.000) with 91.8% sensitivity and 87.5% specificity.CONCLUSION: Circulating miRNA-21 expression and CA 15-3 levels were significantly increased in breast cancer group compared to control group. The miRNA-21 expression increased consistently with breast cancer stage progression. miRNA-21 could serve as superior biomarker compared to CA 15-3.KEYWORDS: biomarker, breast cancer, circulating plasma, liquid biopsy, miRNA-2

Early detection breast cancer: role of circulating plasma miRNA-21 expression as a potential screening biomarker

Background/aim: To explore the potential of the circulating plasma miRNA-21 as an early detection biomarker by comparing earlystage breast cancer (BG) and healthy control (HG) in Indonesian population. Materials and methods: The enlisted patients were 26 adult female early-stage breast cancer patients (stage 1A, 1B, 2A, and 2B) of Airlangga University Hospital from August 2019 to October 2019. Sixteen volunteers were recruited as matching healthy subjects. MiRNA-21 expression was quantified by plasma qRT-PCR. Data analysis performed using IBM SPSS Statistics v.24 (IBM Corp., Armonk, NY, USA). MiRNA-21 cut-off, sensitivity, and specificity were analyzed using receiver operating characteristic (ROC) curve. Results: The study included 26 BG and 16 HG subjects. The miRNA-21 expression in BG group was 3.933 (1.181-11.794) and 0.905 (0.164-4.532) in HG group (4.34 folds; P = 0.001), with 1.66 cut-off (92.3% sensitivity; 81.2% specificity). MiRNA-21 expression separated analysis in HG showed a 0.578 times lower expression in menopause subjects [0.651 (0.164-0.414)], compared to premenopause ones [(1.123 (0.758 - 4.532); P = 0.031]. Yet, in BG group, 1.729 times higher miRNA-21 expression was observed in menopause subjects (6.021 ± 3.583), compared to premenopause ones (3.500 ± 1.517; P = 0.022). Conclusion: Circulating miRNA-21 expression is a potential biomarker for early detection of breast cancer and might act as a breast cancer risk predictor

POTENSI EKSTRAK KENIKIR (Cosmos caudatus Kunth) SEBAGAI AGEN PENGINDUKSI APOPTOSIS SEL PUNCA KANKER PAYUDARA PADA SEL LINE KANKER PAYUDARA SUBTIPE LUMINAL-A

Kanker payudara merupakan kanker dengan prevalensi dan mortalitas terbanyak pada wanita di dunia dan Indonesia. Salah satu penyebab tingginya angka kejadian dan kematian yaitu cancer relapse atau kekambuhan penyakit kanker setelah prosedur terapi. Regimen terapi kanker yang sudah diakui dan diterapkan di dunia seperti kemoterapi, radioterapi, dan pembedahan masih menyisakan bahaya cancer relapse. Cancer relapse diduga disebabkan oleh sel punca kanker (cancer stem cell)yang merupakan kelompok khusus sel kanker yang memiliki sifat seperti sel punca,yaitu kemampuan self-renewal serta diduga berperan dalam resistensi terhadap kemoterapi dan radioterapi. Pengembangan terapi dengan sasaran sel punca kanker berpotensi sebagai konsep pendekatan terapi kanker yang dapat meminimalisir risiko cancer relapse. Beberapa penelitian mengemukakan bahwa bahan herbal yang tersedia di Indonesia memiliki kemampuan untuk menginduksi kematian sel kanker melalui mekanisme apoptosis. Namun penelitian pada sel punca kanker payudara masih membutuhkan pengujian secara lebih lanjut. Kenikir mengandung kandungan flavonoid yang cukup tinggi, seperti quercetin. Quercetin merupakan flavonol yang diduga berperan dalam proses induksi apoptosis sel kanker. Pengujian kemampuan induksi apoptosis sel punca kanker payudara dengan ekstrak kenikir diperlukan untuk memastikan potensinya sebagai kandidat cancer stem cell targeted therapy. Rumusan masalah : 1) apakah pemberian ekstrak kenikir (Cosmos caudatus Kunth)dapat menginduksi kematian sel punca kanker payudara subtipe luminal-A melalui jalur apoptosis?, 2) berapakah dosis optimal pemberian ekstrak kenikir (Cosmos caudatus Kunth) dalam menginduksi apoptosis sel punca kanker payudara subtipe luminal-A? Tujuan umum penelitian ini adalah mengetahui kemampuan induksi apoptosis ekstrak kenikir (Cosmos caudatus Kunth) terhadap sel punca kanker payudara subtipe luminal-A. Tujuan khusus antara lain 1) mengetahui kemampuan ekstrak kenikir (Cosmos caudatus Kunth) dalam menginduksi apoptosis sel punca kanker payudara subtipe luminal-A, 2) mengetahui dosis optimal pemberian ekstrak kenikir (Cosmos cadatus Kunth) dalam menginduksi apoptosis sel punca kanker payudara subtipe luminal-A. Manfaat karya tulis ini ini bagi pengembangan ilmu adalah untuk menjadi referensi dan landasan untuk penelitian di masa mendatang, terutama dalam penelitian mengenai apoptosis, sel kanker, dan potensi kemampuan terapeutik ekstrak tumbuhan kenikir. Diharapkan karya tulis ilmiah ini dapat menambah pengetahuan masyarakat dan menjadi referensi dalam pemanfaatan tumbuhan kenikir dalam masyarakat luas selain sebagai bahan makanan. Penelitian ini terdiri dari tiga tahap : Tahap pertama, ekstraksi dan karakterisasi ekstrak kenikir. Tahap kedua, proses dediferensiasi untuk menghasilkan CSC enriched culture dan karakterisasinya. Tahap ketiga, eksperimen dengan menguji kemampuan ekstrak kenikir dalam mempengaruhi viabilitas CSC dan juga mekanisme kematian sel yang diinduksinya. Hasil dari penelitian tahap pertama, diperoleh ekstrak metanolik daun kenikir dengan konsentrasi flavonoid total sebesar 41,22 mg ± 0.69312 mg/100g daun keniki

Inactivated SARS-CoV-2 vaccine candidate immunization on non-human primate animal model: B-cell and T-cell responses immune evaluation

Background: SARS-CoV-2 vaccine was proven to be an effective and efficient measure for mitigating pandemic. COVID-19 infection and mortality subsided along with the increaseing COVID-19 vaccination coverage. Vaccine and health resource equity are predominant factors in COVID-19 pandemic management. Vaccine development for Indonesia, aims to ensure a sustainable pandemic control and steady national stability restoration. A decent vaccine must induce immunity against COVID-19 with minimum adverse reaction. Immunogenicity and ability to induce neutralizing antibody evaluation needs to be performed as part of the SARS-CoV-2 inactivated vaccine development from East Java, Indonesia isolate (Vaksin Merah Putih-INAVAC). Objective: This research demonstrated INAVAC performance in inducing the production neutralizing antibody along with its effects on CD4+ and CD8+ cells response in Macaca fascicularis (non-human primate). Methods: Two dosages of 3 μg and 5 μg were tested, compared to sham (NaCl 0.9%) in 10 Macaca fascicularis (2 injection intramuscular with 14 days interval). All animals were monitored daily for clinical signs. Nasopharyngeal samples were analyzed using qRT-PCR while the serum were tested using ELISA and neutralization assay, whereas PBMCs were flowcytrometrically analyzed to measure CD4+ and CD8+ population. Results: It is observed that both vaccine doses could stimulate relatively similar immune response and neutralizing antibody (end GMT post challenge = 905,1), whereas higher CD8+ cells response were reported in the 5 μg group after the 3rd day post-challenge. The dose of vaccine that produce adequate immune cell stimulation with neutralizing antibody induction can be adopted to clinical study, as favorable result of these parameters could predict minimum adverse reaction from inflammation response with balanced immune response. Conclusions: Therefore, it is concluded that Vaksin Merah Putih-INAVAC with 3 μg dose showed a favorable potential to be developed and tested as human vaccine

Molecular docking and dynamic simulation of conserved B cell epitope of SARS-CoV-2 glycoprotein Indonesian isolates: an immunoinformatic approach [version 1; peer review: 3 approved]

Background: An immunoinformatic approach may be useful to investigate the conserved region in the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Indonesia isolates. The aim of this study was to investigate Indonesian SARS-CoV-2 isolates based on B cell epitopes by targeting the conserved regions in the spike glycoprotein to trigger increased multi-variant virus neutralization and memory response for the development of vaccine seed candidates. Methods: SARS-CoV-2 spike glycoprotein gene sequences originating from Indonesia were compared with Wuhan (China), the United Kingdom, South Africa, India, the United States, and Brazil isolates obtained from the NCBI and GISAID databases. The recognition of antigens was carried out directly using B cells through the B cell receptor (BCR). An indirect B cell activation by Cluster of Differentiation (CD)4+ T cells and major histocompatibility complex (MHC)-II was predicted through the binding with human leukocyte antigen (HLA) based on IC50 value. In addition, vaccine allergenicity and toxicity were investigated. During the molecular complex examination, the 3D peptide structure was investigated and the lowest amount of energy formed when the vaccine candidate peptide bound to BCR and MHC-II was calculated. Results: As a result, the spike glycoprotein sequences of Indonesian SARS-CoV-2 isolates had conserved regions which were very similar to reference countries such as China, the United Kingdom, South Africa, India, the United States, and Brazil. Conclusion: It was predicted that the conserved regions could be identified as the epitope of B and T CD4+ cells that produced the peptides for vaccine candidate with antigenic, non-allergen, and non-toxic properties

Dedifferentiation of MCF-7 Breast Cancer Continuous Cell Line, Development of Breast Cancer Stem Cells (BCSCs) Enriched Culture and Biomarker Analysis

Abstract BACKGROUND: Cancer stem cells (CSCs) eradication might serve as a robust approach for cancer eradication. MCF-7 as breast cancer continuous cell line is known to contain breast CSCs (BCSCs) for its capability to maintain its original tumor population. CSCs enriched culture is a fundamental tool for CSCs targeted therapy development. Effective and unsophisticated CSCs dedifferentiation protocol for producing CSCs enriched culture is needed. METHODS: MCF-7 cells were cultured initially in Dulbecco's Modified Eagle Medium (DMEM) low glucose medium then changed to DMEM:F12. Serum starvation was performed during each medium refreshment gradually with fetal bovine serum (FBS) concentration of 10%, 5%, 2.5% until reaching 1% FBS concentration. Stable MCF-7 culture was then adapted to serum free culture system, containing DMEM:F12, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and B27 supplement as dedifferentiation protocol for 18 days. Cluster of differentiation (CD)44 and CD24 double staining immunocytochemistry was performed to evaluate cell stemness. RESULTS: The population of cells expressing BCSCs markers (CD44+/CD24low) in non-adherent single cells subpopulation was significantly increased after the dedifferentiation procedure (70.39%) compared to control groups (0.71%) (p<0.05). In contrast, the expression of BCSCs marker in adherent single cells subpopulation and for both adherent and non-adherent mammosphere the BCSCs markers showed a stable expression. CONCLUSION: BCSCs enrichment of breast cancer cell cultures from MCF-7 breast cancer cell line can be performed. Breast cancer cell plasticity is observed during the dedifferentiation protocol. Development of dedifferentiation inducing protocols can serve as an important foundation for breast cancer therapy development through BCSCs elimination

Factors Associated with Absolute Neutrophil Count Dynamics and Docetaxel-Adryamicin-Cyclophosphamide (TAC) Chemotherapy Induced Neutropenia During Extended Filgrastim Administration in Breast Cancer Patients

Background: Myelosuppressive effects of chemotherapy for breast cancer treatment may trigger chemotherapy-induced neutropenia (CIN) and febrile neutropenia (FN). Filgrastim has been widely used as prophylaxis against CIN and FN. However despite filgrastim administration, some study showed FN still occur and cause patient vulnerability to infection. This study aims to evaluate factors associated with Absolute Neutrophil Count (ANC) dynamics and Docetaxel-Adryamicin-Cyclophosphamide (TAC) CIN during extended filgrastim administration in breast cancer patients. Methods: Patients were selected among breast cancer in-patients who fulfilled the eligibility criteria. Patient characteristics data and ANC were collected. The entire patients received 5μg/kg/day filgrastim by subcutaneous injection 24 hours post-chemotherapy. ANC was monitored daily and filgrastim administration was stopped when ANC reached >10000/mm3 or 14 days of administration. Kruskall-Wallis test and Spearman Correlation test was performed to analyze ANC dynamics and CIN-related factors. Results: This study included 42 breast cancer patients. Patient age median was 52 (31-70) years old. ANC nadir could be observed around 5-7 days after chemotherapy and FN occurred in two out of 38 grade 4 neutropenia patients (4.8%). Critical ANC lasted for 1 day, 2 days, and 3 days respectively in 9 (23.7%), 25 (65.8%) and 4 (10.5%) patients. There was no correlation between neutropenia and age. ANC slope and recovery duration did not show a significant difference. However, depth of nadir is inversely correlated with the duration of ANC recovery (>10000/mm3) and the duration during the peak on the 2nd day until reaching nadir both with fair strength, r = −0.489 and r = −0.438 (p <0.05), respectively. No sepsis incidence had manifested. Conclusion: CIN still occured in breast cancer patient receiving filgrastim primary prophylaxis regardless of age and neutropenia severity. Nadir as the lowest point of ANC should be noted as a pivotal milestone for ANC slope and recovery evaluation