Anti-cancer effect of the combination of sodium pentaborate pentahidrate and vitamin d active form calcitriol against hepatocellular carcinoma

Abstract

This invention relates to a formulation comprising a combination of sodium pentaborate pentahydrate and calcitriol, the active form of vitamin D, which shows anti-cancer effect when used in the treatment of hepatocellular carcinoma. The object of the invention is to provide an anti-cancer effect against hepatocellular carcinoma by a formulation obtained from the combination of sodium pentaborate pentahydrate and calcitriol.

Description

[0001] ANTI-CANCER EFFECT OF THE COMBINATION OF SODIUM PENTABORATE PENTAHIDRATE AND VITAMIN D ACTIVE FORM CALCITRIOL AGAINST HEPATOCELLULAR CARCINOMA

[0002] Technical Field

[0003] This invention relates to a formulation comprising a combination of sodium pentaborate pentahydrate and calcitriol, the active form of vitamin D, which shows anti-cancer effect when used in the treatment of hepatocellular carcinoma.

[0004] Prior Art

[0005] Hepatocellular carcinoma (HCC) is the 5th most common type of primary malignant liver cancer of epithelial origin, ranking 3rd in terms of mortality. Chronic liver disease and cirrhosis are the main risk factors for hepatocellular carcinoma. In addition, Hepatitis B and C viruses, excessive alcohol intake, obesity, and diabetes are the leading risk factors worldwide [1]. HCC is generally two to four times more likely to occur in men than in women [2], In 2020, approximately more than 900,000 people were diagnosed with HCC [3]. The disease being asymptomatic in the early stages prolongs the time it takes for patients to be diagnosed and causes late stages diagnoses after cirrhosis. The survival rate after diagnosis ranges from approximately 6 months to 20 months [4],

[0006] Hepatocellular carcinoma treatments are decided based on the Barcelona Clinic Liver Cancer (BCLC) system depending on tumor stages, size, liver functions and expected benefits after treatment [5]. While resection, transplantation and local ablation are preferred for HCC patients in the early stage, TARE method can be preferred for patients in the moderate stage and systemic treatments can be preferred for patients in advanced stages (6). Since 80-90% of cases are diagnosed at a late stage, most of the treatment methods are very limited, and due to insufficient proven studies on tumor recurrence after treatment, recurrence is encountered within 5 years in most HCC patients [7], Therefore, there is still no effective treatment for HCC. Boron is a biologically important element that has been found to affect many mechanisms in animals and plants. Extensive research is being carried out using the boron element in the clinical studies of various diseases. Research has shown that boron accelerates wound healing and is anti-inflammatory [8]. The anti-cancer effects and cancer growth inhibitory properties of different boron derivatives have been proven in many different types of cancer such as hepatocellular and colorectal cancer [9, 10], lung cancer

[0011] , prostate cancer

[0012] , and breast cancer

[0013] . Bortezomib, a boronic acid derivative, is a boron-derived drug approved by the FDA for the first time showing proteasome inhibitory properties

[0014] , The effects of bortezomib on HCC have been shown in studies to be anti-proliferative and pro-apoptotic, however, since HCC can rapidly gain resistance to the drug, future studies focus on combining Bortezomib with different substances and increasing its effect (15). Sodium pentaborate pentahydrate (NaB), a boron derivative, has also been found to have an anti-cancer effect in assays conducted with lung cancer cell lines

[0016] . Furthermore, other studies have shown that NaB has anti-microbial and anti-viral effects

[0017] ,

[0007] Vitamin D is a compound belonging to the group of fat-soluble vitamins, the majority of which can be synthesized in human skin as a result of sun exposure. Some of its most important functions in the body are its relationship with the mechanism of calcium and phosphate

[0018] . The two main forms of vitamin D important for humans are vitamin D2 (ergocalciferol) of plant origin and cholecalciferol, vitamin D3 of animal origin, which is derived from 7-dehydrocholesterol in the skin. The liver has a huge role in the synthesis of vitamin D in the body. Ultraviolet-B rays in the sun rays convert the 7- dehydrocholesterol in the skin into previtamin D3, and then this intermediate quickly forms vitamin D3, depending on the temperature. Since the synthesized vitamin D3 is not biologically active, it is transported to the liver with the help of vitamin D-binding proteins in the blood. Vitamin D3that reaches the liver is converted to 25-(OH)D3, then transported to the kidney and converted into 1 ,25-(OH)2D3 (calcitriol), the active form of vitamin D. This is the main circulating form of vitamin D found in the body [19, 20, 21], In studies on vitamin D, its protective and healing effects against diseases such as cancer, diabetes, heart diseases, immune system, and bone diseases have been observed

[0022] , According to in vitro and in vivo research, vitamin D can reduce the development of tumors and prevent the growth of cancer cells. It also has antiinflammatory, immunomodulator, pro-apoptotic, and anti-angiogenic properties

[0023] . For this reason, cancer research with vitamin D has increased rapidly. When the relationship between HCC and vitamin D was examined, vitamin D deficiency was seen in HCC patients, and its potential therapeutic effects were mentioned (24).

[0008] Combination therapy studies are an important type of cancer treatment that combines two or more therapeutic agents. Co-administration of therapeutic agents with anticancer effects may increase their efficacy compared to the monotherapy approach as they can target cancer pathways in a synergistic manner. In addition to the potential of this approach to reduce drug resistance; it has advantages such as reducing tumor growth, the possibility of metastasis, cancerous cell population, and inducing the mechanism of apoptosis

[0025] .

[0009] Today, there are some chemotherapeutic drugs used for the treatment of hepatocellular carcinoma. These drugs, which have become part of routine treatment, face resistance problems in the course of the disease and do not have a significant positive effect on survival.

[0010] Summary of the Invention

[0011] The object of the invention is to provide an anti-cancer effect against hepatocellular carcinoma by a formulation obtained from the combination of sodium pentaborate pentahydrate and calcitriol, the active form of vitamin D.

[0012] Detailed Description of the Invention

[0013] “Anti-Cancer Effect of the Combination of Sodium Pentaborate Pentahydrate and Vitamin D Active Form Calcitriol Against Hepatocellular Carcinoma” carried out to achieve the object of this invention is shown in the accompanying figures; in these figures:

[0014] Figure 1 - Graphical representations of MTS analysis results of HepG2 cells treated with (A) Sodium pentaborate pentahydrate, (B) treated with calcitriol, and (C) combinations thereof.

[0015] Figure 2 - Graphical representation of the percentages of cell death leading to apoptosis in HepG2 cells treated with 1000 pg / ml sodium pentaborate pentahydrate, 1 pM calcitriol, and combinations thereof as a result of apoptotic cell death. Figure 3 - Graphical representations of gene expression levels of AKT, GADD45A, CDKN1A, MDM2, p21 , p53, MCM2, MCM5, and MCM7 genes in HepG2 cells treated with control, 1000 pg / ml sodium pentaborate pentahydrate, 1 pM calcitriol, and combination thereof by qPCR method.

[0016] Figure 4 - Graphical representation of the (A) Gene Ontology (Biological Process,) (B) Gene Ontology (Cellular Components), (C) Gene Ontology (Molecular Function) analysis results of proteins with statistically increased significant expression in HepG2 cells with the combination treatment of 1000 pg / ml sodium pentaborate pentahydrate and 1 pM calcitriol compared to the control, obtained by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) technique.

[0017] Figure 5 - Graphical representation of the (A) Gene Ontology (Biological Process,) (B) Gene Ontology (Cellular Components), (C) Gene Ontology (Molecular Function), (D) KEGG analysis results of proteins with statistically decreased significant expression in HepG2 cells with the combination treatment of 1000 pg / ml sodium pentaborate pentahydrate and 1 pM calcitriol compared to the control, obtained by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) technique.

[0018] Figure 6 - Schematic representation of protein-protein interactions of proteins with statistically increased and decreased significant expression in HepG2 cells with the combination treatment of 1000 pg / ml sodium pentaborate pentahydrate and 1 pM calcitriol compared to the control, obtained by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) technique. (A) Proteins with significantly increased expression (B) Proteins with significantly decreased expression

[0019] This invention relates to a formulation obtained from a combination comprising sodium pentaborate pentahydrate and calcitriol, the active form of vitamin D, and the scope of the invention addresses the in vitro study of the anti-cancer effect of this formulation when used in the treatment of hepatocellular carcinoma.

[0020] In the experimental studies carried out within the scope of the invention, firstly, human hepatocellular cancer cell line HepG2 (HB-8065, ATCC) cells were treated with 100- 5000 pg / ml NaB, 0.05-1 pM calcitriol (1 ,25(OH)2D3) and the dual combination thereof, and cell viability test was performed for 24, 48, and 72 hours. According to the results of the cell viability assay in 72 hours, HepG2 cells showed 76.7% viability when treated with 1000 pg / ml NaB, and 81.2% viability when treated with 1 pM calcitriol. When HepG2 cells were treated with the combination of 1000 pg / ml NaB and 1 pM calcitriol, it was observed that this dual combination provided a synergistic effect by reducing cell viability by up to 55% (Figure 1). Furthermore, according to the apoptotic cell death assay, it was observed that the formulation of the invention produced a surprising technical effect, with a percentage of apoptotic cell death of approximately 30% when HepG2 cells were treated with the combination of the two compared to treatment with NaB or calcitriol (1 ,25(OH)2D3) alone (Figure 2).

[0021] When examining the gene expression levels of the expression profiles of AKT, GADD45A, CDKN1A, MDM2, p21 , and p53 genes involved in the apoptosis pathway by qPCR method, it was observed that treatment of HepG2 cells with the combination of 1000 pg / ml NaB and 1 pM calcitriol increased the expression of these genes compared to treatment of cells with 1000 pg / ml NaB alone and 1 pM calcitriol alone compared to control (Figure 3). However, it is believed to stop cell division by reducing the expression of MCM2, MCM5, and MCM7 genes involved in DNA replication.

[0022] Protein profiles of HepG2 cells treated with control, 1000 pg / ml NaB, 1 pM calcitriol (1 ,25(OH)2D3) and the dual combinations thereof were revealed by LC-MS / MS method. Table 1 shows the list of proteins with increased expression in HepG2 cells treated with the control and the dual combination of 1000 pg / ml NaB and 1 pM calcitriol, and Table 2 shows the list of proteins with decreased expression when the protein profiles of the control and the dual combination were compared. Compared to the control, the number of proteins with increased expression was 68, and with decreased expression was 217. When functional Gene Ontology and KEGG pathway analyses of proteins with increased and decreased expression were performed, it was determined that the expression of proteins involved in several pathways such as DNA replication, lipid and carbohydrate metabolism decreased along with the proteins in several pathways with decreased expression (Figures 4 and 5). Figure 6 shows the interactions of these proteins with increased (Figure 6A) and decreased (Figure 6B) expression with each other.

[0023] Table 1- List of protein profile with statistically increased significant expression in HepG2 cells with the combination treatment of 1000 pg / ml sodium pentaborate pentahydrate and 1 pM calcitriol compared to the control, obtained by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) technique

[0024] Table 2- List of protein profile with statistically decreased significant expression in HepG2 cells with the combination treatment of 1000 pg / ml sodium pentaborate pentahydrate and 1 pM calcitriol compared to the control, obtained by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) technique

[0025] EXPERIMENTAL STUDIES

[0026] 1. Cell culture

[0027] Human hepatocellular cancer cell line HepG2 (HB-8065, ATCC) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) nutrient medium (with 2 mM L- glutamine, 100 U / ml penicillin, 100 pg / ml streptomycin) comprising heat inactivated 10% FBS in a humidified 5% CO2 atmosphere at 37 °C. With an interval of 3 days, the cells are passaged.

[0028] 2. Cell viability test - Treatment of HepG2 cell line with Sodium pentaborate pentahydrate and calcitriol HepG2 cells were plated in culture medium at 5,000 cells / well in 96-well culture plates (Corning Glasswork, Corning, NY) in DMEM medium comprising 10% FBS and 1% PSA, then treated with sodium pentaborate pentahydrate (NaB: B5H10NaO13) alone, calcitriol (1 ,25(OH)2D3) alone, and a dual combination of NaB and calcitriol. The viability levels of the cells were measured after 24, 48, and 72 hours. Cell viability analysis was measured by 3-(4,5-di-methyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)- 2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) (CellTiter96 AqueousOne Solution; Promega, Southampton, UK) method following the manufacturer's protocol. After 1.5 hours of incubation, the viability level was obtained using ELISA microplate reader (Bio-tek ELx800, USA) instrument measuring at 490 nm.

[0029] 3. Apoptotic cell death

[0030] HepG2 cells were plated in culture medium at 200,000 cells / well in 6-well plates in DMEM medium comprising 10% FBS and 1% PSA. HepG2 cells were treated with 1000 pg / ml NaB, 1 pM calcitriol (1 ,25(OH)2D3) and dual combinations thereof. After 72 hours, floating and sunken cells were collected. The cells are then incubated with Annexin V and PI staining for 15 minutes in accordance with the manufacturer's protocol of the determined Annexin V / FLOUS (Roche) apoptotic cell death kit. All samples were analyzed with a 10,000-cell count using the FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA).

[0031] 4. Gene expression Analysis by qPCR

[0032] Hepatocellular carcinoma cell line HepG2 cells were plated in cell culture medium at 200,000 cells / well in 6-well plates in DMEM medium comprising 10% FBS and 1% PSA. After 72 hours, floating and sunken cells were collected from HepG2 cells treated with 1000 pg / ml NaB, 1 pM calcitriol (1 ,25(OH)2D3), dual combinations thereof, and nothing as control. Total RNA isolation was performed with Qiagen RNeasy Mini kit from HepG2 cells obtained from four different states. After the RNA concentration was measured with Nanodrop 2000 Spectrophotometer (Thermo scientific), cDNA was synthesized with Sensiscript Reverse Transcription PCR Kit in 500 ng of RNA. GeneMark Bio GM SYBR qPCR Kit and CFX96 RT-PCR system (Bio-Rad, USA) were used for the qPCR assay. Primers for the AKT, CDKN1 A, GADD45A, MDM2, P53, and MCM7 genes were designed using the Primer-BLAST program. The GAPDH gene was used as the reference gene. The list of primers is given in Table 3. Gene expression analysis was calculated by 2-AACt method.

[0033] Table 3- Primary sequences of AKT, GADD45A, CDKN1A, MDM2, p21 , p53, MCM2, MCM5, and MCM7 genes

[0034] 5. Protein Profiling with LC-MS / MS HepG2 cells were plated in culture medium at 200,000 cells / well in 6-well plates in DMEM medium comprising 10% FBS and 1% PSA. HepG2 cells treated with control, 1000 pg / ml NaB, 1 pM calcitriol (1 ,25(OH)2D3) and dual combinations thereof were collected after 72 hours. This assay was done in three repetitions. Protein bands separated by SDS-PAGE from four different samples were cut after fixation and staining with colloidal Coomassie Blue G-250, and after in-gel tryptic digestion, peptides were analyzed by LC-MS / MS using an Ultimate 3000 RSCL nanosystem (Dionex, Thermo Fisher Scientific, USA). Xcalibur 4.0 Software (Thermo Fisher Scientific, USA) was used. High Performance Liquid Chromatography (HPLC) was performed using mobile phases A and B and full-scan MS spectra were obtained. MS / MS was performed with the first ten precursor ions and protein identification analysis was performed with the Proteome Discoverer 2.2 program (Thermo Fisher Scientific, USA). Uniprot / Swissprot database was used to identify the proteins.

[0035] 6. Biyoinformatik Analizdrler

[0036] With the protein profile obtained by LC-MS / MS from HepG2 cells treated with control, 1000 pg / ml NaB, 1 pM calcitriol (1 ,25(OH)2D3) and dual combinations thereof, proteins with significantly increased and decreased expression were determined between control and 1000 pg / ml NaB, control and 1 pM calcitriol (1 ,25(OH)2D3), control and 1000 pg / ml NaB and 1 pM calcitriol (1 ,25(OH)2D3) combination.

[0037] Gene Ontology (GO), KEGG pathway analyses and protein-protein interaction analyses were performed in the STRING Database of proteins with significantly increased and decreased expression between the control and the 1000 pg / ml NaB and 1 pM calcitriol (1 ,25(OH)2D3) combination.

[0038] 7. Result and Evaluation

[0039] Human hepatocellular cancer cell line HepG2 (HB-8065, ATCC) cells were used in experimental studies carried out within the scope of the invention. This cell line was treated with 100-5000 pg / ml NaB, 0.05-1 pM calcitriol (1 ,25(OH)2D3) and the dual combination thereof, and cell viability test was performed for 24, 48, and 72 hours. When the cell viability assay was completed after 72 hours, HepG2 cells showed 76.7% viability when treated with 1000 pg / ml NaB, and 81.2% viability when treated with 1 pM calcitriol. When HepG2 cells were treated with the combination of 1000 pg / ml NaB and 1 pM calcitriol, this dual combination reduced cell viability by up to 55% (Figure 1 ). It has been observed that this dual combination stops cell proliferation by providing a synergistic effect. In the results of the apoptotic cell death assay performed after the cell viability test, it was observed that treating HepG2 cells with the combination of the two had a percentage of apoptotic cell death of approximately 30% compared to treatment with NaB or calcitriol (1 ,25(OH)2D3) alone (Figure 2).

[0040] When examining the gene expression levels of the AKT, GADD45A, CDKN1 A, MDM2, p21 , and p53 genes involved in the apoptosis pathway by qPCR method, it was observed that treatment of HepG2 cells with the combination of 1000 pg / ml NaB and 1 pM calcitriol increased the expression of these genes compared to treatment of HepG2 cells with 1000 pg / ml NaB alone and 1 pM calcitriol alone compared to control (Figure 3). Furthermore, it was observed that the gene expression levels of MCM2, MCM5, and MCM7 proteins involved in DNA replication, which stand out in protein expression profiles by the LC-MS / MS method, are decreased. With the decrease in the expression of these genes, it is believed that the combination of 1000 pg / ml NaB and 1 pM calcitriol stops cell division.

[0041] When the protein profiles of HepG2 cells treated with control and dual combinations of 1000 pg / ml NaB and 1 pM calcitriol (1 ,25(OH)2D3) were examined by the LC-MS / MS method, it was found that the expression of proteins in the DNA replication pathway was reduced along with the proteins in several pathways (Figures 4 and 5).

[0042] In summary, this invention shows that the dual combination of NaB and calcitriol exhibits a synergistic anti-proliferative effect on hepatocellular carcinoma HepG2 cells. Furthermore, comparing NaB and calcitriol combination with cells treated with NaB alone or calcitriol alone has shown that treatment with the combination increases apoptosis in HepG2 cells. Decreased expression of MDM2 and AKT genes and increased expression of p53 and CDKN1A genes further supports the increased apoptosis in HepG2 cells with combination treatment. Protein profiling using the LC- MS / MS method showed that proteins with decreased expression were effective in cell replication and proteins involved in cell metabolism stand out (Figures 4 and 5).

[0043] This invention has shown that the combination of NaB and calcitriol has great potential to be an effective alternative drug in hepatocellular carcinoma.

[0044] REFERENCES [1]. Balogh, J., Victor, D., 3rd, Asham, E. H., Burroughs, S. G., Boktour, M.,

[0045] Saharia, A., Li, X., Ghobrial, R. M., & Monsour, H. P., Jr (2016). Hepatocellular carcinoma: a review. Journal of hepatocellular carcinoma, 3, 41-53. https: / / doi.Org / 10.2147 / JHC.S61146

[0046] [2], Bosch, F. X., Ribes, J., Diaz, M., & Cleries, R. (2004). Primary liver cancer: worldwide incidence and trends. Gastroenterology, 127(5), S5-S16.

[0047] [3]. Liu, Y.; Liu, L. Changes in the Epidemiology of Hepatocellular Carcinoma in

[0048] Asia. Cancers 2022, 14, 4473. https: / / doi.Org / 10.3390 / cancersl 4184473 Academic Editor: Al

[0049] [4], McGlynn, K. A., & London, W. T. (2011 ). The global epidemiology of hepatocellular carcinoma: present and future. Clinics in liver disease, 15(2), 223-x. https: / / doi.Org / 10.1016 / i.cld.2O11 .03.006

[0050] [5]. Llovet, J. M., Bru, C., & Bruix, J. (1999). Prognosis of hepatocellular carcinoma: the BCLC staging classification. In Seminars in liver disease (Vol. 19, No. 03, pp. 329-338). © 1999 by Thieme Medical Publishers, Inc..

[0051] [6]. Llovet, J.M., Kelley, R.K., Villanueva, A. et al. Hepatocellular carcinoma. Nat

[0052] Rev Dis Primers 7, 6 (2021 ). https: / / doi.Org / 10.1038 / s41572-020-00240-3

[0053] [7], AKYILDIZ, U., YALQIN, K. S., & TURKAY, F. c. Hepatoselluler Karsinom

[0054] Tedavisi. Fatih Universitesi, Tip Fakultesi, 1 ig Hastahklan Anabilim Dali Gastroenteroloji Bilim Dali, Ankara, Guncel Gastroenteroloji.

[0055] [8]. Dogan, A., Demirci, S., Qaglayan, A.B. et al. Sodium Pentaborate

[0056] Pentahydrate and Pluronic Containing Hydrogel Increases Cutaneous Wound Healing In Vitro and In Vivo. Biol Trace Elem Res 162, 72-79 (2014). https: / / doi.Org / 10.1007 / s12011 -014-0104-7

[0057] [9]. Yang, Z., Liu, S., Zhu, M., Zhang, H., Wang, J., Xu, Q., ... & Zhu, H. (2016).

[0058] PS341 inhibits hepatocellular and colorectal cancer cells through the FOXO3 / CTNNB1 signaling pathway. Scientific reports, 6(1 ), 1-15.

[0059]

[0010] . Omeroglu Ulu, Z, Bolat, ZB and Sahin, F (2022). Integrated transcriptome and in vitro analysis revealed anti-proliferative effect of sodium perborate on hepatocellular carcinoma cells. J. Trace Elem. Med. Biol. 73: 127011.

[0060]

[0011] . Cebeci, E., Yuksel, B., & §ahin, F. (2022). Anti-cancer effect of boron derivatives on small-cell lung cancer. Journal of Trace Elements in Medicine and Biology, 70, 126923.

[0012] , Cui, Y., Winton, M. I., Zhang, Z. F., Rainey, C., Marshall, J., De Kernion, J. B., & Eckhert, C. D. (2004). Dietary boron intake and prostate cancer risk. Oncology reports, 11 (4), 887-892.

[0061]

[0013] .Scorei, R., Ciubar, R., Ciofrangeanu, C. M., Mitran, V., Cimpean, A., & lordachescu, D. (2008). Comparative effects of boric acid and calcium fructoborate on breast cancer cells. Biological trace element research, 122(3), 197-205.

[0062]

[0014] , Dick, L. R., & Fleming, P. Compared to the antimicrobial activity of the blank silicone material against E. (2010). Building on bortezomib: second-generation proteasome inhibitors as anti-cancer therapy. Drug discovery today, 15(5-6), 243-249.

[0063]

[0015] . Huang, I. T., Dhungel, B., Shrestha, R., Bridle, K. R., Crawford, D.,

[0064] Jayachandran, A., & Steel, J. c. (2019). Spotlight on Bortezomib: potential in the treatment of hepatocellular carcinoma. Expert opinion on investigational drugs, 28(1), 7-18. https: / / doi.Org / 10.1080 / 13543784.2019.1551359

[0065]

[0016] . Cebeci, E., Yuksel, B., & §ahin, F. (2022). Anti-cancer effect of boron derivatives on small-cell lung cancer. Journal of Trace Elements in Medicine and Biology, 70, 126923.

[0066]

[0017] , Demirci, S., Kaya, M. S., Dogan, A., Kalay, §., ALTIN, N. O., YARAT, A., ... &

[0067] §ahin, F. (2015). Antibacterial and cytotoxic properties of boron-containing dental composite. Turkish Journal of Biology, 39(3), 417-426.

[0068]

[0018] . Kuittinen, T., Rovio, P., Staff, S., Luukkaala, T., Kallioniemi, A., Grenman, S.,

[0069] ... & Maenpaa, J. (2017). Paclitaxel, carboplatin and 1 , 25-D3 inhibit proliferation of endometrial cancer cells in vitro. Anticancer research, 37(12), 6575-6581 .

[0070]

[0019] , AKKOYUN, H., Bayramoglu, M., Suat, E. K. L N., & Qelebi, F. (2014). D vitamini ve metabolizma igin onemi. Ataturk Universitesi Veteriner Bilimleri Dergisi, 9(3).

[0071]

[0020] . MacLaughlin, J. A., Anderson, R. R., & Holick, M. F. (1982). Spectral character of sunlight modulates photosynthesis of previtamin D3 and its photoisomers in human skin. Science, 216(4549), 1001-1003.

[0072]

[0021] , Christakos, S., Dhawan, P., Verstuyf, A., Verlinden, L., & Carmeliet, G. (2016).

[0073] Vitamin D: metabolism, molecular mechanism of action, and pleiotropic effects. Physiological reviews, 96(1), 365-408.

[0022] . Cranney, A., Horsley, T., O'Donnell, S., Weiler, H., Puil, L., Ooi, D., Atkinson,

[0074] S., Ward, L., Moher, D., Hanley, D., Fang, M., Yazdi, F., Garritty, C., Sampson, M., Barrowman, N., Tsertsvadze, A., & Mamaladze, V. (2007). Effectiveness and safety of vitamin D in relation to bone health. Evidence report / technology assessment, (158), 1-235.

[0075]

[0023] . Feldman, D., Krishnan, A. V., Swami, S., Giovannucci, E., & Feldman, B. J.

[0076] (2014). The role of vitamin D in reducing cancer risk and progression. Nature reviews cancer, 14(5), 342-357.

[0077]

[0024] , Wu, D. B., Wang, M. L., Chen, E. Q., & Tang, H. (2018). New insights into the role of vitamin D in hepatocellular carcinoma. Expert review of gastroenterology & hepatology, 12(3), 287-294.

[0078]

[0025] . Bayat Mokhtari, R., Homayouni, T. S., Baluch, N., Morgatskaya, E., Kumar, S.,

[0079] Das, B., & Yeger, H. (2017). Combination therapy in combating cancer. Oncotarget, 8(23), 38022-38043.

Claims

CLAIMS1. A formulation for the treatment of hepatocellular carcinoma comprising sodium pentaborate pentahydrate (NaB: B5Hi0NaOi3) and calcitriol (1 ,25(OH)2D3), the active form of vitamin D, and inducing apoptotic cell death in human hepatocellular cancer cell line HepG2 (HB-8065, ATCC) cells upon treatment.

2. A formulation according to claim 1 comprising the dual combination of 1000 pg / ml NaB and 1 pM calcitriol.

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