DOI: https://doi.org/10.21203/rs.3.rs-2028419/v1
Cervical cancer is the fourth most common cause of cancer-related death among women globally. Microbial products represent an available source of anticancer drugs. Thus, this study aimed to extract the total protein from Candidaspecies (CanSp) and subsequently investigate its inhibitory effects against human cervical cancer HeLa cells. This study reports the five total protein of the yeast-to-hyphal transition culture of Candida species, which were then evaluated for their inhibitory potential by cell viability, cell apoptosis and nitrite assays against HeLa cells. Furthermore, transcriptional profile of OCT4B gene was determined using quantitative reverse transcription PCR. Total protein of CanSp1-5 were obtained from Candida species. The result of the protein quantitation assay indicated that the CanSp1-5 exhibited total protein values from 93.72 to 155.25 µg/mL and 89.88 to 144.33 µg/mL by Bradford and micro-Kjeldahl methods, respectively. The CanSp1 was most active with a half-maximal inhibitory concentration of 157.11 ± 0.001 μg/mL and half-maximal effective concentration of 102 ± 0.001 μg/mL. The distinct morphological changes of cells were showed a typical apoptosis. Moreover, a reduction in the nitric oxide concentration was observed in the HeLa cells. The expression level of OCT4B gene was significantly down regulated in the HeLa cells treated with CanSp1-5. These findings highlight the importance of investigating microbial products for the accelerated development new anticancer drugs. In addition, OCT4B gene could be probable molecular target of the CanSp1-5 in the HeLa cells.
Among the several deadly diseases, cervical cancer is the fourth most frequently diagnosed cancer type and the fourth most common cause of cancer death among women. The GLOBOCAN report 2020 showed that 604,000 new cases with cervical cancer were diagnosed, and 342,000 deaths occurred (Sung et al. 2021). Human papilloma virus infection is a major cause of cervical cancer. Additional cofactors are likely to be involved in the cervical carcinogenesis, includeing sexual and reproductive health and social and behavioral factors (Sung et al. 2021; Zhang et al. 2020).
Cervical cancer is defined as an uncontrolled development of normal cells on the surface of the cervix to the transformed cells. The progression of disease through cellular events that lead to hyperplasia and dysplasia in situ and finally to invasive cancer has been recognized, but the molecular basis of cervical carcinogenesis has been not been fully explored (Martínez-Rodríguez et al. 2021).
Abnormal expression of a core transcription factor, such as octamer-binding transcription factors 4 (OCT4), sex determining region Y-box 2 (SOX2) and NANOG may contribute to the initiation and progression of specific types of cancer cervical cancer (Baek et al. 2020; Clemente-Periván et al. 2020). OCT4, a member of the Pit-Oct-Unc family of transcription factors, impacts in the maintenance of self-renewal ability and pluripotency of embryonic stem cells (Li et al. 2015; Zhang et al. 2020). The human OCT4 gene, located on chromosome 6p21, consists of five exons and can be generated by alternative splicing into ten different transcripts, OCT4A, OCT4B, OCT4B1, OCT4B2, OCT4B3, OCT4B4, OCT4B5, OCT4C, OCT4C1 and OCT4D (Clemente-Periván et al. 2020; Li et al. 2015; Mehravar and Poursani 2021; Saha et al. 2018; Zhang, Xu et al. 2020). Subcellular localization of OCT4 isoforms in different regions of cell may be correlated with their functions. OCT4 is specifically expressed in the nucleus of embryonic stem cells and plays fundamental roles in the earliest stage of development as well as maintenance of stemness in embryonic stem cells. OCT4B is highly expressed in the cytoplasm, and suggests that it may cooperate with OCT4A to regulate the progression of cervical cancer linked to angiogenesis and epithelial-mesenchymal transition. The biological function of other OCT4 isoforms are less well appreciated (Li et al. 2015; Mehravar and Poursani 2021).
It is widely acknowledged that resistance to chemo/radio-therapies; prolonged treatment, therapeutic effect and prognosis are the area of intense clinical research (Wang et al. 2021; Zaman et al. 2016). Facing increasing challenges in resistance to chemotherapies, rapid evolution of biotechnology is likely to have an important influence on medicine to alleviate the crisis.
Natural products from microorganisms have been widely used for medical therapeutics and may be of great interest in cancer treatment. Among this group of natural products, proteins and peptides are ubiquitous in microorganisms (Karpińsk and Adamczak 2018; Kornienko et al. 2015; Ng 2004; Peymaeei et al. 2020). Recent data revealed that several compounds and enzymes from fungi have anticancer activity (Noman et al. 2021).
Candida yeasts comprise a group of microbes, which exhibits different effects with a significant potential for biotechnological exploitation or opportunistic pathogenic nature (Kieliszek et al. 2017; Varrella et al. 2021). In this article, we described the isolation of total protein from Candida species. The inhibitory effects of total protein of Candida species (CanSp) on human cervical cancer HeLa cells were investigated by cell viability, cell apoptosis and nitrite assays. Subsequently, we elucidate transcriptional profiling of OCT4B in HeLa cells in response to CanSp.
Candida species and growth conditions
The refrence strains of C. albicans ATCC 90028 and C. tropicalis ATCC 750, two clinical isolates of C. albicans (CaSN1 and CaSN2) and a clinical isolate of C. tropicalis (CtSN1) were used in this study. Clinical vaginal isolates of patients with recurrent vulvovaginal candidiasis, were provided by Microbiology Laboratory, Cellular and Molecular Research Center, Yasuj University of Medical Sciences (Iran). Candida species were plated onto CHROMagar™ Candida (CHROMagar Microbiology, Paris, France) and Sabouraud Dextrose Agar (SDA, Difco Laboratories, USA) plates.
Total protein extraction of Candida species
Total protein were extracted from C. albicans ATCC 90028, CaSN1, CaSN2, C. tropicalis ATCC 750 and CtSN1 and described as CanSp1-5, respectively. Briefly, precultures were incubated in yeast extract peptone dextrose (YEPD) broth medium (containing, 20 g/L Bacto peptone, 10 g/L yeast extract, and 20 g/L dextrose) at 30°C for 18 h. The Candida species cell suspension (1–5 × 106 CFU/mL) used to inoculate fresh human serum at 37°C for 18 h. One handred mg of Candida species cells were collected by centrifugation for 5 min at 5000 rpm. The pellet of cells was then washed twice with ice-cold PBS buffer and once with with sterile Milli-Q water. The cells were re-suspended in 500 µL of 7 M urea, 2 M thiourea, 4% CHAPS and 50 mM DTT buffer containing 1x Protease Inhibitor Mix (GE Healthcare Bio-Science AB, Piscataway, MA, USA). After that, the suspension of cells were sonicated in an UP200H ultrasonic processor (Hielscher, Teltow, Germany) at a frequency of 24 kHz, three times, for 15 s, with pauses of 45 s between sonications. The lysates were separated by centrifugation (4°C, 14,000 rpm, 20 min). The lysates and the remaining cell-free supernatant were sterilized by filtering with a Millipore Ultrafree-15 centrifugal filter device (Millipore, Bedford, MA, USA) for desalting and concentration (Fiorini et al. 2016; Staniszewska et al. 2014; Thomas et al. 2006). A simplified representation of the total protein extraction of Candida species is shown in Fig. 1 (created with BioRender.com).
Total protein quantitation assay
The quantitation of total protein of Candida species (CanSp) were determined using Bradford method (Bradford 1976). A standard curve was prepared by using serially diluted bovine serum albumin (BSA) protein concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µg) in a total volume of 100 µL Milli-Q water. Blank samples were prepared using 100 µL of Milli-Q water. Five mL of Bradford protein reagent (100 mg of Coomassie Brilliant Blue was dissolved in 50 mL of 95% ethanol, mixed with 100 mL of 85% orthophosphoric acid and diluted to a final volume of 1 L with Milli-Q water) was added to each tube and then mixed thoroughly with the standard and exteracted solutions (5 µg). The absorbance was measured at 595 nm using a microplate reader (BioTek, ELx800, Winooski, VT, USA) after incubation at room temperature for 10 min in a dark place. The protein concentration was plotted against the corresponding absorbance, resulting in a standard curve used to measure the total protein concentration in unknown samples.
In addition, the protein concentration of CanSp1-5 was determined by the micro-Kjeldahl method. About 1 mg of each CanSp was digested with with 500 mg potassium sulphate and 2 mL of cupric sulphate/ sulphuric acid Kjeldahl digestion solution at 410°C for 3 h. After cooling, Milli-Q water was added to the hydrolysates before neutralization and titration. The nitrogen content was converted into protein concentration using the coefficient factor 6.25 calculated from the standard curve of serially diluted BSA protein concentrations (Wang et al. 2016).
Cell culture
Human HeLa cell line was obtained from the Pasteur Institute of Iran, Tehran, Iran and maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich, St Louis, MO) at 37°C, 5% CO2 and > 90% humidity for 24 h.
Trypan blue dye exclusion assay
Trypan blue exclusion assay was used to determine the number of viable cells present in a cell suspension. A sample of HeLa cells (10 mL) was collected from during the logarithmic growth phase and pipetted into a counting chamber of hemocytometer (HC-B080A Improved Neubauer counting chamber Blood cell counter chamber, Mainland, China). The cell sample was stained 1:1 with 0.4% trypan blue (Sigma) and incubated 3 min at room temperature. Cells excluding trypan blue (viable cells) were counted using phase contrast inverted microscope (Olympus, Corp., Tokyo, Japan) with a hemocytometer (Strober 2015).
Cell viability assay
The HeLa cells with a cell density of 1 × 104 cells/well was seeded in 96-well flat bottom microplates and incubated at 37°C, 5% CO2 and > 90% humidity for 24 h for proper attachment. Each well received increasing concentrations of CanSp1-5 (0, 7.813, 15.625, 31.25, 62.5, 125, 250 and 500 µg/mL) and incubated under the same condition for 24 h. The last two columns were used as controls, i.e., without treatment. The inhibitory effect was assessed using 3-(4, 5-Dimethyl-1, 3-thiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT, Sigma) dye. Then 10 µL of 5 mg/mL MTT solution was added into each well and plates were incubated at 37°C for 4 h in a dark place. Dimethyl sulfoxide (150 µL) was added into each well and mixed thoroughly to dissolve formazan crystals. The absorbance was measured at 570 nm using a microplate reader (BioTek). The inhibitory effect was calculated as a percentage of the untreated control value (Kokhdan et al. 2018).
Determination of IC50 and EC50 concentrations
The half maximal inhibitory concentration (IC50) and half maximal effective concentration (EC50) values were calculated from a standard curve on the basis of linear-regression analyses (Kokhdan et al. 2018; Kuete et al. 2011).
Cell apoptosis assay
Aperoximatly 1 × 105 HeLa cells were added onto a 6-well cell culture plate containing coverslips and incubated at 37°C, 5% CO2 and > 90% humidity for 24 h. Following attachment, cells were treated with treated with CanSp1-5 at concentration equal to IC50 and incubated under the same condition for 24 h. The untreated cells in 1% DMSO were used as a negative control. Olympus phase contrast inverted microscope (Japan) was used to visualize the apoptosis in cells (Kokhdan et al. 2018; Motadi et al. 2020).
Nitrite assay
The effect of CanSp1-5 on free radical nitric oxide (NO) synthesis was measured by the formation of nitrite in culture supernatants according to the Griess method. One hundred microliters of treated cell culture supernatants was incubated with 100 µL of Griess reagent (1% (w/v) sulfanilamide /0.1% (w/v) N-(1-naphtyl) ethylenediaminedihydrochloride (Sigma) in 2.5% (v/v) H3PO4) at room temperature for 10 min. Then, the absorbance was measured at 550 nm using a microplate reader (BioTek). The nitrite concentration was determined with reference to a standard curve of sodium nitrite (µM/mL) (Tezuka et al. 2001).
Quantitative reverse transcription PCR (RT-qPCR)
HeLa cells were treated with CanSp1-5 at concentration equal to IC50, and then total RNA was extracted using Kiazol (Kiazist Life Sciences, Iran) according to the manufacturer’s protocol. The reverse transcription reaction was carried out according to the manual using RevertAid First Strand cDNA synthesis kit (Fermentas, St. Leon-Ro,Germany), 2 µg of total RNA as the template and oligo dT primer. The OCT4B and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were selected to amplification whose primer sequences are listed in Table 1. The qRT-PCR reaction was performed by the reverse transcription samples using a Maxima SYBR Green qPCR (Bio-Rad, Munich, Germany). The qRT-PCR reactions were performed in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) in accordance with the following program: 1. 94°C 7 min; 2. 94°C 30 s; 3. 58°C 30 s; 4. 72°C 1 min; 5. repeat steps 2–4 45 times; melting curve analysis from 65 to 95°C, 0.5°C per 5 s increments. Relative quantitative of transcriptional levels of OCT4B gene was normalized with respect to housekeeping gene (GAPDH) and calculated by the ΔΔCt method (Norouzi et al. 2021).
Target gene |
Primer name |
Sequence (5'-3') |
Product size (bp) |
Reference |
---|---|---|---|---|
Octamer binding transcription factor 4 B |
OCT4B |
F ATGCATGAGTCAGTGAACAG |
303 |
Li et al. 2015 |
R CCACATCGGCCTGTGTATAT |
||||
Glyceraldehyde-3-phosphate dehydrogenase |
GAPDH |
F GAAGGTGAAGGTCGGAGTC |
226 |
Li et al. 2015 |
R GAAGATGGTGATGGGATTTC |
One-way ANOVA was used to analyze data. Tukey’s post hoc comparison was performed to compare gene expressions of the groups (*P < 0.01; **P < 0.001; ***P < 0.0001). All data are expressed as the mean ± S.D. of at least three independent experiments. All analyses were conducted using SPSS 18.0 and GraphPad Prism 7.
Total proteins (CanSp1-5) were extracted from C. albicans ATCC 90028, CaSN1 and CaSN2, and C. tropicalis ATCC 750 and CtSN1, which were interconnected with the yeast-to-hyphal transition under fresh human serum influence after 18 h of incubation at 37°C. The protein concentration was determined by the Bradford and micro-Kjeldahl methods. Figure 2 displays the standard curve, regression equation and the trend line of the standard protein of the BSA. In addition, representative protein concentrations of CanSp1-5 are depicted in Fig. 2. The standard protein curve and regression equations indicated that the CanSp1-5 exhibited total protein values ranging between 93.72 to 155.25 µg/mL and 89.88 to 144.33 µg/mL by Bradford and micro-Kjeldahl methods, respectively.
We assessed the cytotoxic effect of CanSp1-5 downregulation on cell viability using the colorimetric MTT assay (Fig. 3). Following treatment of HeLa cells with varying concentrations of CanSp1-5, the percentage viability of HeLa cells decreased in a dosedependent manner. The IC50 and EC50 values of CanSp1-5 are presented and compared in Table 2. Statistically significant differences between the CanSp1-3 extracted from C. albicans (ATCC 90028, CaSN1 and CaSN2) and CanSp4, 5 from C. tropicalis (ATCC 750 and CtSN1) were observed (P < 0.0001). The IC50 value of CanSp1-3 extracted from C. albicans (ATCC 90028, CaSN1 and CaSN2) was marginally lower when compared with CanSp4, 5 from C. tropicalis (ATCC 750 and CtSN1). Similarly, the impact of CanSp1-5 on HeLa cells were assessed using EC50 value. The CanSp1 was most active with an IC50 of 157.11 ± 0.001 µg/mL. The result of CanSp1 from C. albicans ATCC 90028 showed EC50 in lower concentration of 102 ± 0.001 µg/mL.
Candida species |
CanSp |
IC50 (µg/mL)*** |
EC50 (µg/mL)*** |
---|---|---|---|
C. albicans ATCC 90028 |
CanSp1 |
157.11 ± 0.001 |
102 ± 0.001 |
C. albicans CaSN1 |
CanSp2 |
184.53 ± 0.002 |
120 ± 0.002 |
C. albicans CaSN2 |
CanSp3 |
203.39 ± 0.001 |
132 ± 0.001 |
C. tropicalis ATCC 750 |
CanSp4 |
197.04 ± 0.001 |
138 ± 0.001 |
C. tropicalis CtSN1 |
CanSp5 |
214.93 ± 0.002 |
150 ± 0.002 |
Note. ***P < 0.0001 |
Furthermore, downregulation of CanSp1-5 induced cellular apoptosis in HeLa cells after 24 h of treatment. The HeLa cells treated with CanSp1-5 at concentration equal to IC50 showed distinct morphological changes corresponding to typical apoptosis, including cell detachment from surface, changed from spindle to round shaped, cell shrinkage and membrane rupture (Fig. 4).
However, at concentration equal to IC50, the CanSp1-5 were able to decrease in NO concentration in HeLa cells as compared with the untreated control (Fig. 5).The standard sodium nitrite curve and regression equations revealed that the CanSp1-5 exerted suppressive activity on the NO synthesis with inhibition percentage ranged from 27.9 to 37.8% (P < 0.001).
We carried out a transcriptional profile of OCT4B gene in HeLa cells treated with CanSp1-5. We first used a panel of different CanSp of Candida species at concentration equal to IC50, which allows relative quantitative analyses of OCT4B gene expression. The expression of OCT4B was significantly (P < 0.01 and P < 0.001) down regulated in the HeLa cells treated with CanSp1-5 but not in untreated control. Relative with untreated control, the expression levels of OCT4B were down regulated by 1.60-, 1.22-, 1.18-, 1.58-, and 1.15-fold after 24 h of treatment in HeLa cells treated with CanSp1-5 from C. albicans ATCC 90028, CaSN1 and CaSN2, and C. tropicalis ATCC 750 and CtSN1, respectively. The box plots allow comparison of relative quantitative of transcriptional levels of OCT4B gene at concentration equal to IC50 of CanSp1-5 (Fig. 6).
Owing to the emergence of drug-resistant cervical cancer, it has been an urgency to discovery of new therapeutic agents. The application of natural products from microorganisms in inhibiting cervical cancer is interesting, given their distinctive benefits, including abundance of species of microorganisms, cost effective and fewer toxic side effects (Demain, 2014; Law et al. 2020; Zhang et al. 2021).
Fungi are biotechnologically important group of microorganisms. Fungi are a valuable source for natural product anticancer drug discovery, including aphidicolin, β-glucans, neoechinulin A (Chaichian et al. 2020; Hyde et al. 2019; Kimoto et al. 2007; Yu et al. 2021). In this study, we extracted total protein from clinical isolates of C. albicans and C. tropicalis during yeast to filamentous transition. The ability of Candida species is to grow in several morphological forms such as yeast and a range of filamentous forms that include true hyphae and pseudohyphae. In addition, the yeast to hyphal transition is regulated by many pathways that secretes several enzymes such as proteinases, phospholipases, and hemolysins (Wooten et al. 2021; Zuza-Alves et al. 2017).
Here, we report the cytotoxic activites, apoptosis-inducing effects and NO synthesis inhibition of total protein extracted from Candida species (CanSp1-5) against HeLa cells. In addition, the gene expression profiling of OCT4B was also evaluated in HeLa cells in response to CanSp1-5. Natural product from Candida showed strong activity against cancer. C. albicans β-glucan represent potential anticancer activity (Peymaeei et al. 2020; Suzuki et al. 2021). Thus, the observed cytotoxicity activity reported in this study can be attributed to the specific proteins/enzymes of C. albicans and C. tropicalis such as cell surface-associated proteins and secreted components present in cell-free supernatants especially with respect to the yeast to hyphal transition (Ebanks et al. 2006; Thomas et al. 2006). Ebanks et al. (2006) reported the proteomic analysis of protein profiles during yeast to filamentous transition. A total of 86 unique proteins identified, corresponding to cell surface-associated proteins. Noman et al. (2021) discussed the use of fungal proteins as an effective anti-cancer agent. Among well-known anticancer compounds, producing fungi are Aspergillus and Penicillium species, which have been shown to possess anticancer activity against the human cervical, ovarian, breast, and colon, pancreatic and colorectal cancers. A number of proteins and peptides related to fungi have been discovered from different habitats, which are known to possess anticancer agents (Deshmukh et al. 2018; Li et al. 2018).
He et al. (2020) evaluated the anti-cervical cancer effect of secondary metabolites of Ginkgo biloba. Interestingly, they confirm the high anti-proliferation activity of these secondary metabolites against HeLa cells as well as promote cell apoptosis and blocked cell migration. Moreover, the the special secondary metabolites of G. biloba markedly attenuated the growth of HeLa cells implanted tumor in mice model. Majoumouo et al. (2020) demonstrated that the secondary metabolites of endophytic fungi extracts effectively inhibit the growth of human cervical cancer cells through apoptosis and attenuation of chemoresistance. Furthermore, Mani et al. (2021) found that a natural bioactive secondary metabolite mediated from Curvularia australiensis has the ability to reduce the cervical cancer tumor in an impressive way by fixing the effective dose for anti-inflammation analysis.
In this study, however, the CanSp1-5, which showed cytotoxic activity against HeLa cells, were obviously reduce the expression of the OCT4B gene after 24 h of treatment. The expression of OCT4B has received great attentions during the past years. Since the alternation in OCT4B depended on the progression of cervical cancer and tumorigenesis (Li et al. 2015; Mehravar and Poursani 2021). Several anti-cervical cancer drugs such as fucoxanthin and calcitriol, have been proved to have inhibitory functions to the expression of genes promoting cell proliferation in human HeLa cells (Wang et al. 2014; Ye et al. 2020).
This study suggested that CanSp1-5 might play a significant role in the inhibitory effect on HeLa cells. Moreover, this effect is mediated by downregulation of OCT4B. The OCT4B gene could be probable molecular target of the CanSp1-5 in the HeLa cells. Together, our findings shed new light on the application of CanSp1-5 in the fight against cervical cancer. More studies need to be performed to find protein profiles of CanSp1-5 that could play a major role in the potentially cytotoxic effects against HeLa cells.
Acknowledgements The authors would like to thank the Islamic Azad University of Yasooj. The results presented in this study are part of the Master thesis (16.11.97).
Funding The authors received no external funding for this study.
Data Availability All relevant data can be provided upon request.
Authors’ Contributions F. A. and A.K. conceived the study and supervised the research. E.P.K., M.A. and P.R. performed experiments, and analyzed data. F. A. and A.K. wrote the paper with all other authors. All authors reviewed the manuscript.
Declarations
Conflicts of interest The authors declare that they have no conflicts of interest.
Research involving human participants and/or animals All procedures performed in studies involving human participants, which are obtained from Microbiology Laboratory, Cellular and Molecular Research Center, Yasuj University of Medical Sciences, were in accordance with the ethical standards of the institutional and/or national research committee and with the 2008 Helsinki declaration.
Informed consent The informed consent was provided with patients for the use of their samples in study.
Financial disclosure The authors received no external funding for this study.