|Year : 2020 | Volume
| Issue : 1 | Page : 12-17
Effect of Vitamin D3on nonmelanoma skin cancer cells: A comparative in vitro study
Eva Dohle, Pasinee Vorakulpipat, Sarah Al-Maawi, Rita Schröder, Patrick Booms, Robert Sader, Charles James Kirkpatrick, Shahram Ghanaati
Department of Oral, Cranio-Maxillofacial and Facial Plastic Surgery, Frankfurt Orofacial Regenerative Medicine (FORM) Laboratory, University Hospital Frankfurt Goethe University, Frankfurt am Main, Germany
|Date of Submission||26-Feb-2020|
|Date of Acceptance||18-Mar-2020|
|Date of Web Publication||16-Apr-2020|
Prof. Shahram Ghanaati
Department of Oral, Cranio-Maxillofacial and Facial Plastic Surgery, University Hospital Frankfurt Goethe University, 60590 Frankfurt am Main
Source of Support: None, Conflict of Interest: None
Background: The use of Vitamin D3, as an alternative drug, combined with common therapeutic strategies to treat nonmelanoma skin cancers, has recently attracted attention. However, in vitro data on Vitamin D3action on different tumor cell lines compared to healthy cells are lacking. Aims and Objectives: In this context, the present study aimed to investigate the potential role of Vitamin D3's ability as an antitumor treatment. Materials and Methods: Cell growth, cell viability, and apoptosis as well as cell cycle distribution were comparatively assessed in a squamous cell carcinoma (SCC) cell line, a basal cell carcinoma (BCC) cell line, and healthy primary normal human epidermal keratinocytes (NHEK) in response to various Vitamin D3concentrations. Results: Tumor and healthy cells clearly responded differently to Vitamin D3application with regard to metabolic activity and apoptosis. The application of Vitamin D3reduced the metabolic activity of the BCC and SCC cancer cell lines (and not NHEK) and induced cell cycle arrest. Furthermore, Vitamin D3-mediated increased apoptosis was observed in tumor cells but not in healthy primary keratinocytes. Conclusions: Our findings indicate an antiproliferative and proapoptotic Vitamin D3-dependent effect on skin cancer cells in vitro, highlighting Vitamin D3as a potential and beneficial alternative drug for further studies with respect to possible clinical strategies to treat nonmelanoma skin cancers.
Keywords: Antitumor treatment, basal cell carcinoma, squamous cell carcinoma, Vitamin D3
|How to cite this article:|
Dohle E, Vorakulpipat P, Al-Maawi S, Schröder R, Booms P, Sader R, Kirkpatrick CJ, Ghanaati S. Effect of Vitamin D3on nonmelanoma skin cancer cells: A comparative in vitro study. Int J Growth Factors Stem Cells Dent 2020;3:12-7
|How to cite this URL:|
Dohle E, Vorakulpipat P, Al-Maawi S, Schröder R, Booms P, Sader R, Kirkpatrick CJ, Ghanaati S. Effect of Vitamin D3on nonmelanoma skin cancer cells: A comparative in vitro study. Int J Growth Factors Stem Cells Dent [serial online] 2020 [cited 2021 Jan 22];3:12-7. Available from: https://www.cellsindentistry.org/text.asp?2020/3/1/12/282567
| Introduction|| |
Nonmelanoma skin cancer (NMSC), which comprises the keratinocyte cancers' basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), is the most common form of cancer in Caucasians, with a continuous increase in incidence worldwide., BCC accounts for 75% of all NMSC cases and is a very slow growing and locally invading tumor that generally rarely metastasizes, whereas SCC comprises 25% and shows a higher rate of metastasis. Despite the fact that surgical excision and radiotherapy as well as chemotherapy are still common therapeutic approaches for BCC and SCC, undesirable scarring, excruciating inflammation, drug resistance, and secondary cancers are some of the side effects following these therapies.
The use of alternative drugs of differing actions in combination with common therapeutic strategies to treat NMSC has recently attracted attention. In this context, the anticancer activity of Vitamin D3 has been broadened, as revealed by its cytostatic or cytotoxic effect on abundant cancer cell types as well as on head-and-neck tumors, for example, it was reported to exert antiproliferative effects on a murine BCC cell line and a SCC cell line. In addition, Vitamin D3 also modulates mediators of apoptosis and directly induces apoptosis via caspase activation in human SCC cell lines., Although clinical trials have been designed to evaluate the potential effects of Vitamin D3 on NMSC, identifying the precise role of Vitamin D3 during the formation and progression of human skin cancers is difficult. This is, among other factors, caused by the fact that sun exposure might cause both Vitamin D3 production and skin cancer. Despite the consensus that more is not better, with the recognition of many in vitro studies, an association between the exposure of tumor cells to high concentrations of Vitamin D3 and anticancer effects has been observed.,, Physiological serum levels of Vitamin D3 in humans vary, and a lower serum concentration limit is generally defined as 30 ng/ml. Vitamin D3 is classically known for its potential role in bone formation and phosphate and calcium metabolism. Synthesis of the biologically active form of Vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) (Vitamin D3), involves a multistep process initiated in skin keratinocytes via ultraviolet B radiation. The skin is unique among organs, in that it is capable of equipping all elements essential for the synthesis, activation, and catabolism of Vitamin D3, as well as responding to it via the Vitamin D receptor (VDR).,, Accumulating epidemiological evidence suggests that low Vitamin D3 levels may be multifold and established later in life in the form of increased occurrence of bone fractures and enhanced susceptibility to infection and are accompanied by a higher frequency of numerous cancers in different populations.,
During this study and to investigate the potential role of Vitamin D3's ability as an antitumor treatment, we assessed cell growth, cell viability, and apoptosis and comparatively analyzed cell cycle distribution in SCC, BCC, and healthy primary normal human epidermal keratinocytes (NHEK) in response to various Vitamin D3 concentrations (10 nM, 100 nM, and 1000 nM). The aim of this study was to elucidate whether tumor and healthy cells respond differently to Vitamin D3 and to estimate interrelated Vitamin D3 reply in SCC cells, BCC cells, and NHEK.
| Materials and Methods|| |
Cell culture experiments
The human BCC cell line (BCC-1/KMC) was obtained from Dr. Chia-Yu Chu, National Taiwan University., BCC cells were cultivated in RPMI 1640 medium supplemented with GlutaMAX™ (Thermo Fischer Scientific, Dreieich, Germany), 10% fetal calf serum (FCS) (Biochrom GmbH, Berlin, Germany), and 1% penicillin/streptomycin (P/S) (Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA). The human SCC cell line (SCC-25) was purchased from the German Collection of Microorganisms and Cell Cultures (no. ACC 617, DSMZ, Braunschweig, Germany). SCC cells were cultivated in Dulbecco's MEM nutrient mixture Ham's F12 (Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA) supplemented with 20% FCS and 1% P/S. NHEK isolated from juvenile foreskin were provided by the Clinic for Dermatology, Venerologie and Allergology, University Clinic Frankfurt am Main, Germany. The acquisition and application of the cells that were used in this study were in accordance with the principle of informed consent and approved by the responsible Ethics Commission of the state of Hessen, Germany. NHEKs were grown in serum-free Keratinocyte Growth Medium 2 (PromoCell, Heidelberg, Germany) with Supplement Mix (PromoCell, Heidelberg, Germany). All cells were maintained at 37°C in a humidified atmosphere of 5% CO2.
1α,25-dihydroxyvitamin D3 (Vitamin D3) treatment
1α,25(OH)2D3 (Vitamin D3) was purchased from Sigma Aldrich Chemie GmbH (Sigma-Aldrich, St. Louis, MO, USA) and solubilized in absolute ethanol. A 1 mM stock solution was stored in the dark at −20°C for up to 2 months. Dilution series of Vitamin D3 was made by diluting the stock solution in the appropriate complete medium so that the final ethanol concentration in 1000nM of Vitamin D3 was 0.1%. BCC cells, SCC cells, and NHEK were seeded 24 h prior to Vitamin D3 treatment (10 nM, 100 nM, and 1000 nM), and the cell response to Vitamin D3 was analyzed after 3 h, 48 h, and 72 h of treatment [Figure 1].
|Figure 1: Schematic overview of the experimental setting. (a) Squamous cell carcinoma, basal cell carcinoma, and primary normal human epidermal keratinocytes were plated 24 h prior to Vitamin D3 supplementation and analyzed after 3 h, 48 h, and 72 h of Vitamin D3treatment. (b) Basal cell carcinoma, squamous cell carcinoma, and primary normal human epidermal keratinocytes were treated with 0 nM (control), 10 nM, 100 nM, and 1000 nM Vitamin D3|
Click here to view
Cell viability: MTS assay
Cells were seeded at an appropriate density in each well of a 96-well plate (5000 cells/well for early time points [3 h and 48 h] and 2500 cells/well for 72 h). After 24 h, the cells were treated with different concentrations of Vitamin D3 for the indicated time points (3 h, 48 h, and 72 h). A CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, Wisconsin, USA) was used following the manufacturer's instructions. Briefly, 30 μl of MTS solution was added to each well containing the sample in 150 μl of the culture medium and incubated for 1 h at 37°C before the absorbance was measured at 490 nm with a plate reader (Infinite M200, TECAN, Crailsheim, Germany). Blanks consisted of 150 μl of media with 30 μl of MTS solution incubated on plastic plates. All experiments were performed independently three times, each in triplicate, and statistical significance was defined as*P < 0.05 and **P < 0.01.
DNA quantification assay: Crystal violet staining
DNA was quantified by using crystal violet staining. Briefly, the cells were fixed in methanol-ethanol solution (at a ratio of 1:2) and stored in Dulbecco's phosphate-buffered saline (DPBS) at 4°C for up to 1 week. After washing three times with DPBS, the cells were incubated with 0.1% crystal violet solution (in aquadest) at room temperature (70 rpm, 20 min). After the incubation period, the unbound dye was washed out with tap water, and the plates were thoroughly air-dried overnight. The color crystals on the plates were eluted with 100 μl/well of 33% acetic acid in water (Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA) for 15 min at room temperature (70 rpm), and absorbance was measured with the use of a plate reader (Infinite M200, TECAN, Grödig, Austria) at a wavelength of 560 nm. The cell number is expressed as a function of the optical density at 560 nm of the sample in relation to the untreated control. Acetic acid incubated on plastic was used as a blank sample. All experiments were performed independently three times, each in triplicate. The percentage of metabolic activity [Figure 2] was calculated as the ratio of cell viability (MTS) to the relative total viable cell count, as measured by the crystal violet assay.
|Figure 2: Metabolic activity (%) in response to Vitamin D3treatment in basal cell carcinoma, squamous cell carcinoma, and primary normal human epidermal keratinocytes after 3 h (a and a'), 48 h (b and b'), and 72 h (c and c') of Vitamin D3application. Metabolic activity was calculated by determining cell viability via the MTS assay and normalized to the relative total viable cell count, as measured by crystal violet staining and quantification. a-c provides an overview of metabolic activity in response to all applied Vitamin D3concentrations, whereas a'-c' highlights the effect of 1000 nM Vitamin D3on cellular metabolic activity. Statistical significance was defined as *P < 0.05 and **P < 0.01|
Click here to view
Cell viability assay: Determination of dead cells
To evaluate the amount of dead cells in response to Vitamin D3 treatment, BCC cells, SCC cells, and NHEK were seeded into 6-well plates and treated with Vitamin D3 as previously described. After the treatment periods (3 h, 48 h, and 72 h), floating and adherent cells were harvested with 0.25% trypsin-EDTA (BCC, SCC) or 0.04% trypsin-EDTA (NHEK) and transferred to flow cytometry analysis tubes tubes (4 × 10 cells/ml). The cells were washed once by adding 1 ml of CellWASH (BD Biosciences, Franklin Lakes, NJ, USA), centrifuged at 500 × g for 5 min, and resuspended in 500 μl of CellWASH (BD Biosciences, Franklin Lakes, NJ, USA). Then, 2.5 μl of propidium iodide (PI) solution (1.0 mg/ml in water: Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA) was added directly to the sample and protected from light. Flow cytometric analysis was performed on a FACSCalibur flow cytometer (Becton Dickinson Bioscience, Franklin Lakes, USA) using a 488-nm laser beam, and detection was performed using a 585/15 bandwidth filter. A total of 10,000 events were acquired for each condition. The number of viable and dead cells was calculated with CellQuest Pro software (Becton Dickinson, Franklin Lakes, USA). Experiments were repeated three times, and statistical significance was defined as *P < 0.05 and **P < 0.01.
Cell cycle analysis
The cell cycle was analyzed by quantifying DNA content by flow cytometry as described previously. Briefly, the cells were seeded into 6-well plates and treated with 1000 nM Vitamin D3. After 48 h of treatment, the cells were harvested and fixed in 70% ethanol for 2 h at 4°C and then stored for up to 1 week at 4°C. Before analysis, the cells were washed with PBS, centrifuged, and resuspended in 1 ml of PI staining solution (0.1% [v/v] Triton X-100, 10 μg/ml PI [Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA]). The cells were incubated for 30 min at room temperature and protected from light. The DNA content of 10,000 cells was used to evaluate cell cycle distribution using a FACSCalibur flow cytometer (Becton Dickinson Bioscience). The results were analyzed by Flowing Software version 2.5.1 (Becton Dickinson Bioscience) and are depicted as the percentage of cells with DNA content corresponding to cells in G0/G1, S, and G2/M phases of the cycle. All experiments were independently performed in triplicate.
For the comparison of mean values between two groups, the multiple comparison t-test was used to calculate a P value. Statistical significance was defined as follows: *P < 0.05, ** P < 0.01, and ***P < 0.001. The results are presented as the mean ± standard error of the mean and were analyzed by using MS Excel (Microsoft Office, Microsoft) and GraphPad Prism 6 software version 6.0C (La Jolla, CA, USA) to produce graphic images and to complete statistical analyses.
| Results|| |
Effect of Vitamin D3 on the metabolic activity of basal cell carcinoma cells, squamous cell carcinoma cells, and primary normal human epidermal keratinocytes
To analyze the metabolic activity of BCC cells, SCC cells, and NHEK in response to treatment with Vitamin D3 at different concentrations, an MTS assay was performed after 3 h, 48 h, and 72 h to assess cell viability, and the data were normalized to the relative total viable cell count as measured by crystal violet staining and quantification [Figure 2]. [Figure 2]a,[Figure 2]b,[Figure 2]c provides an overview of the comparison of the metabolic activity of all tested cell types in response to different concentrations of Vitamin D3 calculated after the different time points, whereas [Figure 2]a'[Figure 2]b,[Figure 2]c' highlights the response of BCC cells, SCC cells, and NHEK to a high concentration of Vitamin D3 (1000 nM) after 3 h (A'), 48 h (B'), and 72 h (C'), representing the most substantial and cell-differing Vitamin D3 effect with regard to the metabolic activity of the cells. After 3 h of treatment, highly concentrated Vitamin D3 application (1000 nM) led to a significant decrease in metabolic activity in all the three cell types after 3 h of treatment [Figure 2]a' compared to untreated control cells. In general, the metabolic activity of BCC and SCC cells in response to different Vitamin D3 concentrations exhibited a similar pattern independent of the applied concentration and analyzed time point [Figure 2]a,[Figure 2]b,[Figure 2]c, which was characterized by a decrease in metabolic activity after all the three analyzed time points of Vitamin D3 application and in response to various Vitamin D3 concentrations. In contrast, the metabolic activity of NHEK decreased after 3 h of Vitamin D3 treatment but increased after 48 h and 72 h in a concentration-dependent manner [Figure 2]b and [Figure 2]c. Remarkably, Vitamin D3 seems to have a clearly positive effect on NHEK when treated with high doses (1000 nM), indicated by an increase in metabolic activity [Figure 2]b' and [Figure 2]c'. This positive effect was not observed when BCC or SCC cells were treated with Vitamin D3 at high concentrations, in contrast to a decrease in metabolic activity in response to Vitamin D3 application, as observed after 72 h of treatment [Figure 2]c'. This difference in cell type-dependent responses to Vitamin D3 with regard to metabolic activity can be assessed as statistically significant after 3 h and 72 h of treatment [Figure 2]a' and [Figure 2]c'. Although the same trend was also observed after 48 h of treatment, this trend was not statistically significant [Figure 2]b'.
Determination of the apoptosis of basal cell carcinoma cells, squamous cell carcinoma cells, and primary normal human epidermal keratinocytes in response to Vitamin D3 treatment
To examine whether cell death might contribute to Vitamin D3 application, the total amount of dead cells was assessed via flow cytometry analysis after staining with the fluorescent intercalating agent PI in response to the treatment of cells with 10 nM, 100 nM, and 1000 nM Vitamin D3 for the indicated time points (3 h, 48 h, and 72 h), and the results are depicted in [Figure 3]. Highly concentrated Vitamin D3 treatment (1000 nM) to BCC cells significantly increased the amount of dead cells after each analyzed time point compared to untreated controls and compared to lower Vitamin D3 concentrations [Figure 3]a. Although the same trend was observed at each time point, statistical significance was reached only at the second and third time points [Figure 3]a. This increase in the amount of dead cells in BCC cells when treated with 1000 nM Vitamin D3 becomes even more clear when considering [Figure 3]a', which reflects the significant enhancement of apoptotic cells in the course of Vitamin D3 application (1000 nM) from 3 h to 72 h. This effect was not observed for SCC cells to the same extent [Figure 3]b. After 48 h of Vitamin D3 treatment at a high concentration (1000 nM), the number of apoptotic cells was significantly higher than the cells treated with lower concentrations and compared to the control SCC cells. The same pattern was slightly detected after 3 h and 72 h of treatment but was not considered statistically significant. Vitamin D3 application in NHEK resulted in an increase in the amount of dead cells after 3 h, independent of the applied concentration [Figure 3]c. Interestingly, after 48 h and 72 h, the amount of dead cells clearly decreased to very low percentages in all the tested groups, confirming that the amount of dead cells seems to be generally unaffected by Vitamin D3 application after 48 h and 72 h of treatment [Figure 3]c and c'.
|Figure 3: Apoptotic cells in response to Vitamin D3treatment. The percentage of dead cells was determined via propidium iodide staining and quantification in response to treatment with different Vitamin D3concentrations. (a and a') Percentage of dead basal cell carcinoma in response to Vitamin D3application. (b and b') Percentage of dead squamous cell carcinoma in response to Vitamin D3treatment. (c and c') Percentage of dead primary normal human epidermal keratinocytes in response to Vitamin D3application. Statistical significance was defined as *P < 0.05 and **P < 0.01|
Click here to view
Effect of Vitamin D3 on cell cycle distribution in basal cell carcinoma, squamous cell carcinoma, and primary normal human epidermal keratinocytes
As cell cycle arrest may explain a decrease in the metabolic activity of the cells and to elucidate the potential mechanisms for the antiproliferative effects of Vitamin D3 on BCC and SCC cells, cell cycle distribution was studied in response to Vitamin D3 treatment [Figure 4]. Therefore, PI was used as the DNA dye, which binds in direct proportion to the total amount of DNA in the different cell types and with regard to Vitamin D3 treatment. According to their total DNA content evaluated via flow cytometry, appropriate cells were scaled to the different stages of the cell cycle (G0/G1, S, and G2/M). After 48 h of high-concentration (1000 nM) Vitamin D3 treatment, the total number of S phase cells decreased in response to Vitamin D3 in all the analyzed cell types [Figure 4]a and [Figure 4]b. This Vitamin D3-mediated effect on the amount of S phase cells was marginal in NHEK (17.88% vs. 16.15% S-phase cells) compared to BCC (17.9% vs. 12.14% S-phase cells) and SCC (19.37% vs. 12.23% S-phase cells), in which the decrease in the amount of S-phase cells was more distinct [Figure 4]a. This result corresponds to an increased amount of cells in the G0/G1 phase in both BCC (63.27% vs. 71.66%) and SCC (62.89% vs. 71.81%) cells, whereas cells in the G2/M phase only slightly differed in response to Vitamin D3 [Figure 4]a and [Figure 4]b. In general, the distribution of cells according to cell cycle phase differed markedly between the primary cell type (NHEK) and the analyzed cell lines, revealing more NHEK in G2/M phase and less in G0/G1 phase compared to the untreated cells [Figure 4]b. In general, in NHEK, Vitamin D3 application affected cell cycle distribution to a lesser extent, in which the difference in the amount of G0/G1 cells was not notable (56.18% vs. 55.10%).
|Figure 4: Cell cycle distribution in response to Vitamin D3treatment (a and b). For cell cycle distribution analyses, propidium iodide was used as the DNA dye, which binds in direct proportion to the total amount of DNA in the different cell types. According to their total DNA content evaluated via flow cytometry, appropriate cells were scaled to the different stages of the cell cycle (G0/G1, S, and G2/M) after 48 h of treating basal cell carcinoma, squamous cell carcinoma, and primary normal human epidermal keratinocytes with 1000 nM Vitamin D3|
Click here to view
| Discussion|| |
A substantial amount of preclinical data has suggested that Vitamin D3 (1α,25(OH)2D3), also known as calcitriol, acts as an anticancer agent by exerting antiproliferative, antiangiogenic, and proapoptotic effects in numerous cancer models of SCC and breast and lung cancers as well as prostate adenocarcinoma.,,,,,,, Although the antiproliferative activities of Vitamin D3 against a number of solid tumor cell lines, i.e., human colon cell lines and human prostate cell lines, have already been reported, studies have not examined the effect of Vitamin D3 in a SCC cell line and a BCC cell line compared to NHEK. During this study, SCC, BCC, and NHEK were treated with different concentrations of Vitamin D3 for different time points, and the behavior of the cancer cell lines was compared to that of healthy cells with regard to metabolic activity, apoptosis, and cell cycle distribution in response to Vitamin D3 application. Vitamin D3 treatment led to a significant decrease in metabolic activity in SCC and BCC after 3 h, 48 h, and 72 h of treatment. However, the metabolic activity of NHEK also decreased after 3 h of Vitamin D3 treatment but increased again after 48 h and 72 h in a concentration-dependent manner. Remarkably, we revealed that Vitamin D3 seems to have a clearly positive effect on the metabolic activity of NHEK and a negative effect on the metabolic activity of tumor cell lines when treated with 1000 nM Vitamin D3. McElwain et al. reported an antiproliferative effect of Vitamin D3 on different squamous carcinoma cell lines in vitro, inhibiting cell growth in different SCC models when treated with Vitamin D3 in a dose-dependent manner. As cell cycle arrest might be responsible for a decrease in the metabolic activity of cells, cell cycle distribution was studied in response to Vitamin D3 treatment during this study. Cell cycle interference might be related to Vitamin D3-mediated antiproliferative activity in tumor cell lines, as treatment of SCC cells with 1α,25(OH)2D3 might induce G0/G1 cell cycle arrest. It has been shown that Vitamin D3 might have numerous effects on regulating the cell cycle, and the precise molecular basis is not fully understood. Our findings indicate that Vitamin D3 treatment clearly affects cell cycle distribution in BCC and SCC, shifting S-phase cells in favor of G0/G1 after 48 h of treatment with 1000 nM Vitamin D3. The application of Vitamin D3 results in a decrease in the amount of cells in the S phase of the cell cycle and an increase in the amount of cells in the G0/G1 phase in the SCC and BCC tumor cell lines, indicating the induction of cell cycle arrest in SCC and BCC in response to Vitamin D3 and therefore preventing cells from replicating. The fact that Vitamin D3 application does not considerably affect metabolic activity or the cell cycle distribution of healthy NHEK compared to BCC and SCC, might further support the administration of Vitamin D3 as a potential and beneficial agent for antitumor treatment and should be further analyzed with regard to anticancer research. Although the detailed mechanisms and regulatory machinery were not analyzed in detail during this study, it has already been documented that treatment of SCC with Vitamin D3 induces G0/G1 cell cycle arrest via the regulation of cyclins and their association with cyclin-dependent kinases and cyclin-dependent kinase inhibitors.,, Recent findings also reported the indirect effects of Vitamin D3 on cell cycle regulation and the existence of Vitamin D3 cross-talk with multiple other pathways, i.e., the transforming growth factor-β and phosphatidylinositol 3-kinase signaling cascades., Therefore, our findings thus support performing further in vitro studies that are currently under investigation, analyzing the detailed underlying mechanisms of the antiproliferative effect of Vitamin D3 on BCC and SCC. In addition to the antiproliferative effect of Vitamin D3 on BCC and SCC, we also investigated apoptosis in response to Vitamin D3 application. Our results revealed that cell death was significantly induced in BCC and SCC when Vitamin D3 was supplemented, as indicated by a significant increase in the percentage of dead cells depending on the employed dose of Vitamin D3 in BCC and SCC after 48 h and after 72 h, showing the highest rate of apoptotic cells when 1000 nM Vitamin D3 was applied. Although apoptosis was also induced via Vitamin D3 in NHEK after 3 h of treatment compared to untreated controls, the percentage of dead cells normalized after 48 h and 72 h, and no difference in apoptosis was detected regardless of the Vitamin D3 concentration used. The apoptotic effect of Vitamin D3 on SCC and BCC might be due to the downregulating key mediators of apoptosis, as already reported in numerous studies., Therefore, Vitamin D3 seems to exert its antitumor effects by repressing the expression of antiapoptotic factors or by inducing the expression of proapoptotic factors, finally leading to apoptosis.
| Conclusion|| |
The present comparative study provides in vitro preclinical data supporting the use of Vitamin D3 to increase the therapeutic efficacy in treating SCC and BCC, as we showed that the application of Vitamin D3 reduces the metabolic activity of BCC and SCC cancer cell lines by the induction of cell cycle arrest and leads to a Vitamin D3-mediated increase in apoptosis compared to healthy primary keratinocytes. Therefore, the combination of commonly applied treatments to NMSCs, such as surgical excision, radiotherapy, and chemotherapy, with the addition of Vitamin D3, as an alternative drug, might attract attention for NMSC therapies.
The authors would like to thank Mrs. Verena Hoffman for excellent technical assistance.
Financial support and sponsorship
Roche Pharma AG (EPGZ268304) and the Clinic for Oral, Cranio-Maxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University Frankfurt am Main, supported this work.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Alam M, Nanda S, Mittal BB, Kim NA, Yoo S. The use of brachytherapy in the treatment of nonmelanoma skin cancer: A review. J Am Acad Dermatol 2011;65:377-88.
Griffin LL, Ali FR, Lear JT. Non-melanoma skin cancer. Clin Med (Lond) 2016;16:62-5.
Guix B, Finestres F, Tello J, Palma C, Martinez A, Guix J, et al
. Treatment of skin carcinomas of the face by high-dose-rate brachytherapy and custom-made surface molds. Int J Radiat Oncol Biol Phys 2000;47:95-102.
Sharma P, Saxena S, Aggarwal P. Trends in the epidemiology of oral squamous cell carcinoma in Western UP: An institutional study. Indian J Dent Res 2010;21:316-9.
] [Full text]
Hansen CM, Binderup L, Hamberg KJ, Carlberg C. Vitamin D and cancer: Effects of 1,25(OH) 2D3 and its analogs on growth control and tumorigenesis. Front Biosci 2001;6:D820-48.
Satake K, Takagi E, Ishii A, Kato Y, Imagawa Y, Kimura Y, et al
. Anti-tumor effect of vitamin A and D on head and neck squamous cell carcinoma. Auris Nasus Laryn×2003;30:403-12.
Tang JY, Xiao TZ, Oda Y, Chang KS, Shpall E, Wu A, et al
. Vitamin D3 inhibits hedgehog signaling and proliferation in murine Basal cell carcinomas. Cancer Prev Res (Phila) 2011;4:744-51.
Eisman JA, Macintyre I, Martin TJ, Frampton RJ, King RJ. Normal and malignant breast tissue is a target organ for 1,25-(0H) 2 vitamin D3. Clin Endocrinol (Oxf) 1980;13:267-72.
Eisman JA, Martin TJ, MacIntyre I. Presence of 1,25-dihydroxy vitamin D receptor in normal and abnormal breast tissue. Prog Biochem Pharmacol 1980;17:143-50.
Trump DL, Deeb KK, Johnson CS. Vitamin D: Considerations in the continued development as an agent for cancer prevention and therapy. Cancer J 2010;16:1-9.
Holick MF. Vitamin D deficiency. N Engl J Med 2007;357:266-81.
Laird E, Ward M, McSorley E, Strain JJ, Wallace J. Vitamin D and bone health: Potential mechanisms. Nutrients 2010;2:693-724.
Bikle DD. Vitamin D and the skin: Physiology and pathophysiology. Rev Endocr Metab Disord 2012;13:3-19.
Wierzbicka J, Piotrowska A, Żmijewski MA. The renaissance of vitamin D. Acta Biochim Pol 2014;61:679-86.
Thacher TD, Clarke BL. Vitamin D insufficiency. Mayo Clin Proc 2011;86:50-60.
Fleet JC, DeSmet M, Johnson R, Li Y. Vitamin D and cancer: A review of molecular mechanisms. Biochem J 2012;441:61-76.
Lomas A, Leonardi-Bee J, Bath-Hextall F. A systematic review of worldwide incidence of nonmelanoma skin cancer. Br J Dermatol 2012;166:1069-80.
Pozarowski P, Darzynkiewicz Z. Analysis of cell cycle by flow cytometry. Methods Mol Biol 2004;281:301-11.
Liu M, Lee MH, Cohen M, Bommakanti M, Freedman LP. Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937. Genes Dev 1996;10:142-53.
Mantell DJ, Owens PE, Bundred NJ, Mawer EB, Canfield AE. 1alpha, 25-dihydroxyvitamin D (3) inhibits angiogenesis in vitro
and in vivo
. Circ Res 2000;87:214-20.
Ylikomi T, Laaksi I, Lou YR, Martikainen P, Miettinen S, Pennanen P, et al
. Antiproliferative action of vitamin D. Vitam Horm 2002;64:357-406.
Deeb KK, Trump DL, Johnson CS. Vitamin D signalling pathways in cancer: Potential for anticancer therapeutics. Nat Rev Cancer 2007;7:684-700.
Simboli-Campbell M, Narvaez CJ, Tenniswood M, Welsh J. 1,25-Dihydroxyvitamin D3 induces morphological and biochemical markers of apoptosis in MCF-7 breast cancer cells. J Steroid Biochem Mol Biol 1996;58:367-76.
Colston KW, Chander SK, Mackay AG, Coombes RC. Effects of synthetic vitamin D analogues on breast cancer cell proliferation in vivo
and in vitro
. Biochem Pharmacol 1992;44:693-702.
Getzenberg RH, Light BW, Lapco PE, Konety BR, Nangia AK, Acierno JS, et al
. Vitamin D inhibition of prostate adenocarcinoma growth and metastasis in the Dunning rat prostate model system. Urology 1997;50:999-1006.
McElwain MC, Modzelewski RA, Yu WD, Russell DM, Johnson CS. Vitamin D: An antiproliferative agent with potential for therapy of squamous cell carcinoma. Am J Otolaryngol 1997;18:293-8.
Shabahang M, Buras RR, Davoodi F, Schumaker LM, Nauta RJ, Evans SR. 1,25-Dihydroxyvitamin D3 receptor as a marker of human colon carcinoma cell line differentiation and growth inhibition. Cancer Res 1993;53:3712-8.
Corder EH, Guess HA, Hulka BS, Friedman GD, Sadler M, Vollmer RT, et al
. Vitamin D and prostate cancer: a prediagnostic study with stored sera. Cancer Epidemiol Biomarkers Prev 1993;2:467-72.
Hershberger PA, Modzelewski RA, Shurin ZR, Rueger RM, Trump DL, Johnson CS. 1,25-Dihydroxycholecalciferol (1,25-D3) inhibits the growth of squamous cell carcinoma and down-modulates p21(Waf1/Cip1) in vitro
and in vivo
. Cancer Res 1999;59:2644-9.
Verlinden L, Verstuyf A, Convents R, Marcelis S, Van Camp M, Bouillon R. Action of 1,25(OH) 2D3 on the cell cycle genes, cyclin D1, p21 and p27 in MCF-7 cells. Mol Cell Endocrinol 1998;142:57-65.
Jensen SS, Madsen MW, Lukas J, Binderup L, Bartek J. Inhibitory effects of 1alpha, 25-dihydroxyvitamin D (3) on the G (1)-S phase-controlling machinery. Mol Endocrinol 2001;15:1370-80.
Hmama Z, Nandan D, Sly L, Knutson KL, Herrera-Velit P, Reiner NE. 1alpha, 25-dihydroxyvitamin D (3)-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex. J Exp Med 1999;190:1583-94.
Yanagisawa J, Yanagi Y, Masuhiro Y, Suzawa M, Watanabe M, Kashiwagi K, et al
. Convergence of transforming growth factor-beta and vitamin D signaling pathways on SMAD transcriptional coactivators. Science 1999;283:1317-21.
McGuire TF, Trump DL, Johnson CS. Vitamin D (3)-induced apoptosis of murine squamous cell carcinoma cells. Selective induction of caspase-dependent MEK cleavage and up-regulation of MEKK-1. J Biol Chem 2001;276:26365-73.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]