|Ahead of print publication
Calcitriol potentially alters HeLa cell viability via inhibition of autophagy
Iwan Setiawan1, Ronny Lesmana1, Hanna Goenawan2, Dodi Suardi3, Vesara Ardhe Gatera4, Rizky Abdulah4, Raden Tina Dewi Judistiani5, Unang Supratman6, Budi Setiabudiawan7
1 Departement of Biomedical Science, Physiology Division, Faculty of Medicine; Physiology Molecular, Biological Activity Division, Central Laboratory; Vitamin D Centre, Faculty of Medicine, Universitas Padjadjaran, Bandung, West Java, Indonesia
2 Departement of Biomedical Science, Physiology Division, Faculty of Medicine; Physiology Molecular, Biological Activity Division, Central Laboratory, Universitas Padjadjaran, Bandung, West Java, Indonesia
3 Departement of Obstetrics and Gynecology, Division of Oncology and Gynecology, Faculty of Medicine, Hasan Sadikin Hospital - Universitas Padjadjaran, Bandung, West Java, Indonesia
4 Departement of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Bandung, West Java, Indonesia
5 Vitamin D Centre; Departement of Public Health, Faculty of Medicine, Universitas Padjadjaran, Bandung, West Java, Indonesia
6 Physiology Molecular, Biological Activity Division, Central Laboratory; Departement of Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Bandung, West Java, Indonesia
7 Vitamin D Centre; Departement of Child Health, Faculty of Medicine, Universitas Padjadjaran, Bandung, West Java, Indonesia
|Date of Submission||09-Apr-2020|
|Date of Decision||28-May-2020|
|Date of Acceptance||16-Jul-2020|
|Date of Web Publication||13-Jan-2021|
Departement of Biomedical Sciences, Faculty of Medicine, Universitas Padjadjaran, Bandung, West Java, 45363; Physiology Molecular, Biological Activity Division, Central laboratory, Bandung, West Java, Indonesia, 45363. Vitamin D Centre, Faculty of Medicine, Bandung, West Java, 45363
Source of Support: None, Conflict of Interest: None
Objective: This study was conducted to evaluate the effect of Calcitriol on cellular death in HeLa cells via autophagy and turn over due to mitochondria homeostasis.
Methods: HeLa cell lines were grown in 24-well plates and treated with Calcitriol at varying doses (0.013 μM-0.325 μM) for varying time periods (2, 6, 12, and 18 h). Cell proteins were extracted with scrapers and lysed using RIPA buffer. Western blots were performed for proteins involved with autophagy (Lc3, p62), signaling (mTOR, PI3K, HIF1α), mitochondria (PGC1α, COX4, and Tom 20), and apoptosis (Caspase 3, Caspase 9, and PARP). Protein carbonyl levels were determined by measuring the indirect ROS level. An inhibition study using L-mimosine was performed to analyze the significance of HIF1α.
Results: Calcitriol treatment induced cytotoxicity in a dose- and time-dependent manner and caused growth arrest in HeLa cells. The PI3K-AKT-mTOR pathway was activated, leading to inhibition of autophagy and alterations in mitochondria biogenesis homeostasis. Treatment with Calcitriol produced protein carbonyl levels similar to those in the cisplatin-treated and control groups. Increased ROS levels may cause toxicity and induce cell death specifically in cancer cells but not in normal cells. The inhibition of HIF1α partially rescued the HeLa cells from the toxic effects of Calcitriol treatment.
Conclusion: We suggest that Calcitriol may shut down mitochondrial homeostasis in HeLa cells by inducing the PI3K-AKT-mTOR pathway and inhibiting autophagy, which leads to cell death.
Keywords: Autophagy, cancer, cervix, cholecalciferol, hela, mitochondrial homeostasis
|How to cite this URL:|
Setiawan I, Lesmana R, Goenawan H, Suardi D, Gatera VA, Abdulah R, Judistiani RT, Supratman U, Setiabudiawan B. Calcitriol potentially alters HeLa cell viability via inhibition of autophagy. J Can Res Ther [Epub ahead of print] [cited 2021 Jan 17]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=306944
| > Introduction|| |
Cervical cancer is the second leading cause of death globally, and it is responsible for an estimated 9.6 million deaths in 2018. The Indonesian Ministry of Health stated that the prevalence of cancer in Indonesia in 2018 was approximately 1.8%. Furthermore, a study from GLOBOCAN showed that cervical cancer will become the most frequent cancer in Indonesia, with an estimated 58.000 new cases in 2018. Previous data from the pathology unit at a hospital in Indonesia approximate the cervical cancer incidence at approximately 13/10,000 women, and the mortality rate approximately 7/100,000.
Cervical cancer is seventh in worldwide frequency but ranks the second among women, next only to breast cancer; it is the leading cause of cancer-related death in developing countries. Cervical cancer continues to be a widespread public health problem in women throughout the world, especially in developing countries such as Indonesia. Data from thirteen pathology centers in Indonesia show that cervical cancer ranks the first in frequency among all cancers (23.43% of all people; 31.0% of women with the 10 most common cancers suffer from cervical cancer). Data from various academic hospitals in 2007 showed that uterine cancer is the most common gynecologic malignancy, followed by cancers of the ovary, cervix, vulva, and vagina.
Tobing et al. 2014 reported that Indonesia has a high cervical cancer incidence due to high parity and a high rate of youth marriage. Therefore, finding new approaches for cancer treatment that are easier, less toxic, more efficient, and more economical is an important target. In Indonesia, cervical cancer management outside the context of research is still based entirely on surgery, radiation therapy, and systemic treatment with chemotherapy or hormones. However, despite improvement in cervical cancer therapy, hormonal therapy and chemotherapy, breast cancer remains the leading cause of death from cancer in women. Therefore, there is an urgent need to develop novel and innovative treatment options with excellent clinical efficacy.
A variety of approaches to modulate the tumor microenvironment to increase CAR-T cell anti-tumor activity are currently being researched. A combination of CAR-T cell therapy with existing immunomodulator agents might be beneficial. One of the prospective agents is Vitamin D, which is the precursor to the potent steroid hormone Calcitriol (1,25 [OH] 2D3). The classical role of Calcitriol is to regulate the metabolism of calcium and phosphate, which are essential for bone remodeling. Recent studies suggest that Calcitriol has a role in the immune system. Calcitriol could dramatically change the surface expression of homing receptors on T-cells, resulting in an increased migration ability to sites of infection. Calcitriol is also known to suppress tumor-promoting inflammation. Furthermore, Calcitriol has the potential to affect cancer development and growth by regulating multiple signaling pathways involved in the proliferation, apoptosis, invasion, angiogenesis, and metastasis of cancer cells.
For several decades, researchers have been trying to discover the best treatment for cervical cancer. However, the underlying mechanism for the activity of cervical cancer is not fully understood.,, In addition, there are few available treatments that are nontoxic to normal cells but potent and powerful enough to kill cancerous ones., Clinical patient data showed that low Calcitriol has tight correlation with low survival rate and increased malignancy of cancer. Calcitriol also plays a role in cancer frequency, development, and death., Therefore, understanding the role of Calcitriol-induced cancer cell death is important as a foundation for new cervical cancer treatments.
In this study, by comprehensively evaluating antitumor activity of Calcitriol in cervical cancer cell lines, we generate a new prospective and novel strategy for the management of cervical cancer.
| > Methods|| |
Antibodies were procured from Cell Signaling Technology (LC3 Antibody #2775, SQSTM1/p62 Antibody #5114, mTOR (7C10) Rabbit mAb #2983, COX IV (D73D12) XP® Rabbit mAb #4661, Tom20 (C54G1) XP® Rabbit mAb#3205, HIF1A (D6J9 M) XP® Rabbit mAb #11998, Caspase 3 (49D7) Rabbit mAb #2708, Caspase 9 (Thr389) (108D2) Rabbit mAb #9234, PARP #9542 Rabbit mAb; GAPDH (D16H11) XP® Rabbit mAb #5174, Mitochondrial Marker Antibody Sampler Kit #8674. Vitamin D3 activated 7-dehydrocholesterol Calcitriol (catalog # PHR1237) was purchased from Sigma-Aldrich (St. Louis, MO). HeLa cells were obtained from American Type Culture Collection (ATCC; Manassas, Virginia, USA).
HeLa cells were grown to the appropriate density (usually 70%) in a humidified chamber at 37°C with 5% CO2. The HeLa cells were grown in Dulbecco's modified Eagle medium with 10% fetal bovine serum and 1% penicillin-streptomycin. For Calcitriol experiments, the cells were plated in a 12-well plate and treated with Calcitriol at varying doses for varying lengths of time. Control cells were treated with medium only.
After treatment for 24 h, cells were lysed using RIPA lysis buffer. Immunoblotting was then performed as per the manufacturer's guidelines (Bio-Rad, Hercules, CA, USA) using the protein extraction reagent RIPA Lysis buffer (20 mM Tris-HCl (pH 7.5); 150 mM NaCl; 1 mM Na2EDTA; 1 mM EGTA; 1% NP-40; 1% sodium deoxycholate; 2.5 mM sodium pyrophosphate; 1 mM β-glycerophosphate; 1 mM Na3VO4; 1 μg/ml leupeptin) with added protease inhibitors and 1 mM sodium orthovanadate (a phosphatase inhibitor). The tissue lysates were separated by centrifugation for 15 min at 15,000 xg and 4°C. Protein concentrations in the supernatant were measured through Bradford protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein were resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with the antibody of [Table 1] overnight at 4°C. The antigen-antibody complexes were detected by chemiluminescence with an ECL system (GE Healthcare, Buckinghamshire, UK) and visualized with a LICOR C-DiGiT (USA). The intensity of the bands was quantified using image analysis software (LICOR, C-DiGiT). The blots were reprobed with an anti-GAPDH antibody (1:1000) (Cell Signaling Technology, Danvers, MA) to monitor the quantity and integrity of the protein.
|Table 1: List of primary antibodies for immunoblotting (IB) and immunofluorescence (IFA)|
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Cell structures were examined using an Olympus microscope, and the cell destruction was recorded by using ×200 and ×400 magnification (EVOS XL Core Imaging System, ThermoFisher Scientific, USA).
Protein carbonyl assessment
HeLa cells were cultured by using 5 × 103 cells and were grown to the appropriate density, usually 70%, in 24-well plates; the cells were maintained in a humidified chamber at 37°C with 5% CO2. The cells were divided into three groups: Media (negative control), 50 μM cisplatin, or 0.1 μM vitamin D; the cells were incubated with the treatment for 18 h. Carbonyl content was determined by the derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine, thus leading to the formation of stable dinitrophenyl hydrazone adducts, which can be detected spectrophotometrically at 375 nm and produce a value proportional to the carbonyl present. All samples and standards were run in duplicate. Samples were prepared in a suitable lysis buffer and centrifuged to remove any insoluble material. Samples were diluted with water to a specified protein concentration (Protein Carbonyl Content Assay Kit Sigma, Catalog Number: MAK094).
HIF 1 α inhibition study
HeLa cells were cultured by using 5 × 103 cells and were grown to the appropriate density, usually 70%, in 24-well plates; the cells were maintained in a humidified chamber at 37°C with 5% CO2. The cells were divided into six groups: Negative control, cisplatin, 50 μM Vitamin D, α-mimosine, α-mimosine with 50 μM Vitamin D.
Individual culture experiments were performed in duplicate or triplicate and repeated three times using matched controls; the data from these experiments were pooled. The results were expressed as average mean ± standard error of the mean. The statistical significance of differences was performed using a one-way ANOVA followed by a post hoc Bonferroni test (comparison among three or more groups). P < 0.05 was considered statistically significant.
All experimental procedures were approved prior the study by the Ethical Committee of Universitas Padjadjaran no 959/UN6. KEP/EC/2019.
| > Results|| |
Calcitriol induce HeLa cell death
To assess the Calcitriol effect on HeLa cell viability, we examined HeLa cell with MTS assay. Calcitriol was used in different doses started from 0.02 to 2.6 μg/ml with Cisplatin were used as positive control. Cells viability was examined after 18 h of treatment. MTS assay showed that HeLa cells viability was significantly reduce with increasing dose of Calcitriol [Figure 1]a and [Figure 1]b. High dose Calcitriol (0.33–2.5 μg/ml) induced HeLa cells death, comparable with Cisplatin-treated cell. Then, we observed the changes HeLa cells morphology in dose-dependent and time-dependent manner after Calcitriol treatment [Figure 1]c and [Figure 1]d. HeLa cells morphology was changed in the presence of Calcitriol in a dose-dependent manner [Figure 1]c. Reduction of HeLa cells proliferation was observed in 0.25 and 0.5 μM Calcitriol. In [Figure 1]d, we shown that Calcitriol induce cell death after 18 h. To confirm the effect of Calcitriol on another cell line, we examined the viability of MCF7 cells. Similar results were observed in Calcitriol cytotoxic effect on MCF7 using MTS assay [Supplemental Figure 1]. However, MCF7 cells viability were reduced in lower dose of Calcitriol (0.07 μg/ml) while higher dose of Calcitriol were needed to suppress HeLa cells viability (0.13 μg/ml).
|Figure 1: Calcitriol decreases HeLa cell viability leading to cellular death in dose- MTS plate cell treated by Calcitriol dose dependent (a); Average mean data of MTS are plotted in bar graph for reflecting the cell viability in dependent manner. (b) Disruption HeLa cell morphology in dose - (c) and time- dependent manner (d); **Significant compare to cisplatin group|
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Calcitriol-modulated autophagy and HIF1α
We investigated whether Calcitriol induced HeLa cell death with different dose. We observed that groups HeLa in time dependent and dose-dependent manner. We evaluated the Calcitriol affected autophagy pathway. Treated HeLa cells were subjected to the measurement of LC3 (Atg8) and p62 protein levels. Autophagy-related protein, p62 levels were increased 4 fold compared to the Cisplatin group. It indicated that autophagy was inhibited in Calcitriol treated group [Figure 2]a and [Figure 2]b. In addition, we observed that Calcitriol induced HIF1α protein expression, the activation of which is crucial for inducing cancer cell death. We found that treatment with the HIF1α inhibitor, 50 μM L-mimosine, at least partly inhibits Calcitriol-induced cell death, as shown in [Figure 2]c.
|Figure 2: Time-dependent (a) and dose-dependent (b) treatment of Calcitriol inhibits autophagy and stimulates HIF1α protein level in HeLa Cell. (c) Treatment of 50 μM L-mimmosine rescue and inhibit Calcitriol-induced cell death. Average mean with standard deviation is presented in the graph. Dose-dependent treatment of Calcitriol significantly reduces reduction of mitochondrial protein levels in HeLa Cell (d) Representative blott; (e) Average mean with standard deviation is presented in the graph. *p<0,05; **p<0,01|
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Calcitriol significantly reduced mitochondrial protein levels in HeLa cells
To examine whether Calcitriol alters the mitochondrial number and function before inducing cell death, we treated cells with varying doses of Calcitriol and found that, compared to cisplatin, Calcitriol reduced the levels of the mitochondrial proteins COX IV and Tom 20. Intriguingly, PGC1α levels also increased; however, the levels were not high enough to stimulate mitochondrial biogenesis, as the COX IV and Tom 20 levels remained too low [Figure 2]d and [Figure 2]e.
Calcitriol potentially stimulated mitochondrial dysfunction
To measure the effect of Calcitriol on mitochondrial dysfunction, protein carbonyl levels were measured. We observed that Calcitriol increased protein carbonyl levels almost three-fold when compared to the control and led to autophagy inhibition and decrease of mitochondrial protein levels in HeLa cells. There were the changes in cell morphology after Calcitriol treatment, especially in the structure and integrity of the cell [Figure 3]a, [Figure 3]b, yc, [Figure 3]d.
|Figure 3: Calcitriol significantly induces the protein carbonyl levels and match with autophagy inhibition and decrease of mitochondrial protein levels in HeLa Cell. (a) Cell morphology after calcitriol treatment; (b) level of protein carbonyl; (c) representative blott; (d) average mean with standard deviation is presented in the graph. dose dependent of calcitriol does not stimulate caspase 3, caspase 9, and parp activation in hela cell. (e) representative blott; (f) average mean intensity with standard error mean is presented in the graph. *p<0,05; **p<0,01|
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Calcitriol did not stimulate caspase 3, caspase 9 or PARP, thus leading to apoptosis at a low dose of calcitriol
Death in HeLa cells was induced by apoptosis signaling. Our study showed that low dose Calcitriol (0.013 and 0.13 μM) activated caspase 3, caspase 9 expression. In contrast, high dose Calcitriol did not induce apoptosis signaling. Next, we examine whether activation apoptosis signaling correlated with the changes of PARP expression. Cisplatin induced cleavage of PARP and stimulated apoptotic signaling; however, in Calcitriol treatment, cell death occurred without activating PARP or stimulating apoptosis signaling. Apoptosis was induced only at the high concentrations of Calcitriol [Figure 3]e and [Figure 3]f.
Calcitriol stimulated the PI3K-AKT-mTOR pathway
By exploring the inhibition effect of Calcitriol on autophagy, we measured PI3K, AKT, and mTOR protein level. We found that Calcitriol-treated HeLa cells expressed higher PI3K-AKT-mTOR protein levels significantly. Calcitriol inhibited autophagy via activation of PI3K, AKT, and mTOR protein levels in HeLa cells. In contrast, cisplatin decreased PI3K, AKT, and mTOR protein levels [Figure 4]a and [Figure 4]b. The proposed mechanism can be found in [Figure 4]c.
|Figure 4: Dose-dependent of Calcitriol significantly induces activation of PI3K, AKT and mTOR protein levels in HeLa cell. (a) representative blott; (b) average mean with standard deviation is presented in a graph; (c) proposed scheme of Calcitriol action in HeLa Cell. *p<0,05; **p<0,01|
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| > Discussion|| |
Cervical cancer is the most common cancer in women and the second most common cause of cancer death. In 2010, more than 200,000 invasive cervical cancers were diagnosed in women in the United States, and approximately 40,000 women died of this disease, making it the leading cause of cancer-related death in women. The causes of cervical cancer are not yet completely understood.
Over the past two decades, it has become clear that Calcitriol has many extraskeletal functions, such as immunomodulation and anti-cancer activities., Several reports indicate that Calcitriol deficiency raises the risk of developing cancer and worsens the outcomes for this disease.,, High Vitamin D Receptor (VDR) expression in breast cancer is associated with a reduced risk of death from cancer and an improved prognosis. The biological actions of Calcitriol are mediated by VDR, mostly via genomic action. Calcitriol binds to VDR, thereby causing it to dimerize with retinoid X receptor; this complex then binds to Vitamin D response elements at multiple regulatory regions located at promoters and distal sites of target genes. Calcitriol regulates multiple signaling pathways involved in proliferation, apoptosis, differentiation, inflammation, invasion, angiogenesis, and metastasis, and it therefore has the potential to affect cancer development and growth. Calcitriol has also been reported to regulate microRNA expression, and it may affect cancer stem cell biology. Özgü E et al. 2016 reported that deficiency of Calcitriol and its metabolites is a possible reason for HPV DNA persistence and related cervical intraepithelial neoplasia. Calcitriol correlates with survival rate and malignancy of cervical cancer cells, and the development of Calcitriol treatments and Calcitriol analogs are potentially as important as developing preventative and therapeutic anticancer agents.
Cervical tissue may be a new target organ for therapeutically applied Calcitriol analogs for several reasons, including that VDR is upregulated at the protein level in cervical carcinomas compared with normal cervical tissue and that upregulation of VDR in cervical carcinoma is not exclusively induced by alterations in epithelial differentiation or proliferation but, rather, by different, unknown mechanisms. Therefore, Calcitriol and new Calcitriol analogs exerting fewer calcemic side effects may be promising new drugs for the treatment or chemoprevention of metastasizing cervical carcinomas as well as cervical precancerous lesions.
Consistent with previous reports, Calcitriol (1α,25[OH]2D3) has demonstrated anticancer activity in a variety of cancers. We observed that Calcitriol induces cell death in HeLa cells in a time- and dose-dependent manner [Figure 1]; a similar effect is seen in glioblastoma primary cultures and breast cancer cell lines (unpublished data). It has also been reported that cell death through stimulation of FN1 regulates the viability, apoptosis, migration, invasion, and adhesion of cervical cancer cells through the FAK signaling pathway and is a potential therapeutic target in the treatment of cervical cancer. Altering EAG1 levels via Calcitriol treatment is a potential therapy for cervical cancer;; furthermore, by decreasing HCCR-1 expression and increasing p21 expression, Calcitriol may inhibit HeLa S3 cell proliferation. Many possible mechanisms exist by which Calcitriol could induce cell death in cervical cancer cells.
In the present study, we found that mitochondrial dysfunction may be an alternative pathway for Calcitriol-induced cell death. This hypothesis is supported by the fact that mitochondria are important for the survival of cervical cancer cells. Mitochondrial physiology is strongly regulated by Ca2+. Mitochondria import Ca2+ through the Ca2+ uniporter, which is energized by an electrochemical gradient. Mitochondrial-associated endoplasmic reticulum membranes (MAMs) bring the endoplasmic reticulum type 3 inositol triphosphate receptor (IP3R) Ca2+ release channels into juxtaposition with the mitochondrial Ca2+ uniporter. When not active, the promyelocytic leukemia (PML) protein, found within MAMs, leads to the AKT-mediated hyperphosphorylation of the IP3R3 channels and reduced Ca2+ flux. Because excessive Ca2+ uptake by mitochondria can activate the mtPTP and initiate apoptosis, inactivation of PML may limit apoptosis and increase cancer cell survival. Therefore, homeostasis of mitochondrial biogenesis and mitochondrial degradation are driven partly by autophagy, a process by which cytoplasmic organelles are catabolized. Mitochondrial autophagy is induced by hypoxia, a process which requires the hypoxia-dependent factor-1-dependent expression of BNIP3 and the constitutive expression of Beclin-1 and Atg5, which are both present in cells subjected to prolonged hypoxia.
Interestingly, we found that autophagy is inhibited in both time- and dose-dependent manners upon Calcitriol treatment, whereas the inhibition of autophagy is reflected from the increase or decrease of LC3II (ATG8) represent of autophasome formation and increase of SQTM1 (p62) [Figure 2]. Modulation of p62 is a key of understanding of autophagy activity; p62/SQSTM1 binds directly to LC3 and GABARAP family proteins via a specific sequence motif and link ubiquitinated proteins to the autophagic machinery to enable their degradation in the lysosome. p62 is accumulated when autophagy is inhibited, and decreased levels can be observed. Autophagy is essential for mitochondrial biogenesis, and its inhibition may play a role in cell death of the cancer cell; however, it is not the only mechanism that is involved in cancer cell death. Interestingly, autophagy showed a different manner caspase signaling stimulation. At early time points, our cisplatin control showed the induction of PARP cleavage, which is a process not seen with Calcitriol treatment, thus providing evidence for a new potential mechanism in which Calcitriol induces apoptotic signaling after longer incubation periods.
We also observed a significant decrease in mitochondrial marker proteins, such as Tom20 and COX IV. In normal regulation, decreased mitochondrial function or number will be marked by an increase in PGC1α levels and the initiation of mitochondrial biogenesis. We did not observe any induction in cisplatin treatment. We found a significant increase in PGC1α protein levels; however, mitochondrial protein markers COX IV and Tom20 did not increase [Figure 2]. Mitochondrial autophagy is an adaptive metabolic response that is necessary to prevent the increased levels of reactive oxygen species and cell death. Consistent with previous data, we demonstrate that Calcitriol significantly increases the protein carbonyl levels and inhibits autophagy activity, which may disrupt mitochondrial functions [Figure 4]. Interestingly, VD-induced cell death via HIF 1α inhibition. Cotreatment with or without L-mimosine, an HIF1α inhibitor, significantly decreased the capability of Calcitriol to stimulate cell cancer death [Figure 2].
HIF-1 controls the transcription of hundreds of genes in response to hypoxia and represses mitochondrial biogenesis and respiration. Interference with the HIF-1-dependent regulation of mitochondrial respiration under the conditions of prolonged hypoxia (≥24 h) leads to increased ROS levels and increased apoptosis.,,,, The transcription factor HIF1 induces glycolysis under low oxygen conditions through the upregulation of genes encoding glucose transporters, glycolytic proteins and angiogenic factors (such as erythropoietin and vascular endothelial growth factor) and the inhibition of mitochondrial function via induction of PDHK1, thereby inhibiting PDH and slowing the conversion of pyruvate to mitochondrial acetyl-CoA.
Mitochondria also regulate HIF1α through a process in which mitochondrial ROS from complex III inactivates PHD2 and thus stabilizes HIF1α. Mitochondrial Sirtuin 3 (SIRT3) also modulates HIF1α through mitochondrially generated ROS. There has been little study regarding the mechanism by which Calcitriol affects cancer cells, especially cervical cancer cells. Our study reveals that there is potential induction of P13K, mTOR, AKT, and HIFα plays a central role in Calcitriol-induced cell death, which is not similar to the pattern seen in cisplatin-induced cell death [Figure 3]. Unfortunately, we could not clarify the role of AKT-mTOR activation or deactivation in our study, since we did not run the phosphosrylation of AKT and mTOR. We also found that HIF 1α protein expression is significantly stimulated by Calcitriol and that it plays a central role in initiating Calcitriol-induced cell death [Figure 2].
We suggest Calcitriol may shut down mitochondrial homeostasis in HeLa cells by inducing the PI3K-AKT-mTOR pathway and inhibiting autophagy, which leads to cell death [Figure 4]. It leads to an increase of ROS to toxic levels, which may induce cancer cell death. Taken together, Calcitriol supplementation may potentially exert a direct anti-cancer effect on cancer cells and on the cancer microenvironment. We believe that our study generates new perspectives and novel strategies for the management of cervical cancer.
Financial support and sponsorship
This study was funded by Indonesia Ministry Education and Culture in Penelitian Dasar Unggulan Perguruan Tinggi (PDUPT) scheme no. 3851/UN.6/KUP/2019 to Budi Setiabudiawan and Ronny Lesmana.
Conflicts of interest
There are no conflicts of interest.
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