The comparison of curcumin and nanocurcumin effects on the expression of E6 and E7 human papilloma virus oncogenes and P53 and pRb factors in HeLa and broblast cell lines

Cervical cancer is a common cancer in women around the world. Human papillomavirus infection is main cause of this cancer. Currently treatment methods include chemotherapy, radiation therapy and surgery. These methods usually cause severe reactions and drug resistance in the patients, so nding a safe natural drug for treatment can be very helpful. Curcumin is a compound derived from the root of the Curcuma longa plant and is the active ingredient in a food spice. Various studies have conrmed the anticancer, chemoprotective and genes and proteins regulatory effects of this component. Insolubility, low cellular uptake and low bioavailability are common limitations of curcumin use. To overcome these limitations, a nano-formulation of curcumin has been developed in our laboratory. In this study, the effect of nanocurcumin on cellular apoptosis induction of Hela cancer cells was compared with curcumin. It has demonstrated that nanocurcumin function is three time more effective than curcumin in same concentration. This nano-formulation of curcumin in 15µM concentration was able to reduce the expression of E6 and E7 oncogenes, and increase P53 and Rb tumor suppressors in HeLa cancerous cells with p-value = 0.001 − 0.0001, whereas curcumin altered these genes expression in 50µM concentration with p-value = 0.05 − 0.01. Nanocurcumin has no signicant effect on the viability of normal broblast cells. Therefore, this compound has no effect on normal cells. These ndings are consistent with the results of previous studies about nanocurcurcumin in other types of cancer. Therefore, nanocurcumin may be a suitable option to consider for cervical cancer treatment.


Introduction
Cervical cancer is the third most common cancer in women. 569,000 new cases and 311,000 deaths from this cancer were reported in 2018 [1]. Important risk factors for this cancer include human papillomavirus (HPV), smoking, and immune disorders [2]. HPV is the most critical factor in the development of cervical cancer and HPV test is performing for screening of cervical cancer [3,4].
Carcinogenic HPV types (such as HPV16 and HPV18) target cervical epithelial cells and are responsible for almost all cases of cervical cancer [5].
The human papillomavirus genome can encode eight genes include L1, L2, E1, E2, E4, E5, E6 and E7 [6]. Among these genes, E6 and E7 are the main oncogenic factors of this virus. These factors target different cellular proteins. E6 protein can bind to P53 tumor suppressor and inactive it. E7 protein can disrupt the pRb, terminates their association with the E2F transcription factor family and destroys cell cycle regularity [7]. In the vast majority of human tumors, the functions of pRb and p53 are impaired.
Decreased level of Rb gene leads to disruption of cell proliferation regulation and apoptosis. P53 gene reduction also eliminates the sensitivity of cells to checkpoint and apoptosis signals [8]. In cervical cancer, the expression of E6 and E7 genes increase in the full-thickness epithelial lesion [9,10]. In this study, the effect of nanocurcumin on the E7, Rb and P53 expression in RNA and protein levels was investigated in HeLa cancer cell line. HeLa cells are immortal cells taken from a woman with cervical cancer. HPV infection in these cells is an important cause of cancer progression. HPV virus genome can integrate into the host cell genome and express viral oncogenes. Two HPV oncogenes, E6 and E7, are expressed in cervical carcinomas and carcinoma-derived cell lines such as HeLa.
Curcumin, a safe and well-known natural compound, has attracted the attention of researches. This compound is derived from the rhizomes of the Curcuma longa and use as nutritional supplement. Curcumin affects many of cellular pathways. So far, various applications of this compound have been reported, such as inducing cell differentiation [11,12] and anti-cancer potential by regulating many proliferative, oncogenic, and chemo-resistance associated genes or proteins. [13]. The effect of curcumin on cervical cancer cells and its anti-HIV property have been determined [14], but nano-formulation of curcumin containing OA400 nano-carrier has not been evaluated in cervical cancer so far. Curcumin can arrest the proliferation of cervical cancer cells depending on the time and concentration and it is more active in HPV-infected cells. The anti-cancer activity of curcumin against cervical cells was due to regulation of telomerase activity, Ras and ERK signaling pathways, cyclin D1, COX-2 and iNOS activity and mitochondrial pathway. Recent proteomic studies have shown that curcumin causes signi cant changes in proteins associated with cell metabolism, cell cycle, and carcinogenesis in HeLa cells. The decreasing of cervical tumor volume has also been demonstrated in the curcumin-treated mouse model [15].
Due to the fact that void curcumin has low bioviability, low cellular absorption and insolubility in water, a nano-formulation of curcumin was developed in Professor Sadeghizadeh laboratory to overcome these limitations. A nano-carrier contain oleic acid and PEG400 was used in this carrier [16].
In this study, the reduction of cell viability and apoptosis induction were determined in HeLa cells that treated with nanocurcumin. Interestingly these results were not observed for broblast cells. Gene and protein expression analysis were shown the upregulation of P53 and pRb factors and downregulation of E6 and E7 under the in uence of nanocurcumin treatment. These data indicate the potential of nanocurcumin to consider as an ameliorative agent in cervical cancer investigations.

Nanocurcumin preparation
The method of preparation of nanocurcumin has been stated in previous studies in the prof.
Sadeghizadeh laboratory [17]. Brie y, OA400 nanocarrier was fabricated during the estri cation reaction between oleyl chloride (purchased from SigmaAldrich Company) and polyethylene glycol 400 (Merck Company) in the presence of triethylamine (Merck Company) and chloroform (Merck Company) as the solvent at 25 C. Appropriate concentrations of curcumin (purchased from SigmaAldrich Company) and nanocarrier (1:25 w/w) were mixed together in order to nanocurcumin preparation.

Cell culture and reagents
The human cervical cancer cell line (HeLa cell line RRID:CVCL_003) and broblast normal cells were purchased from the Pasteur Institute of Iran. DMEM growth medium contains 10% (v:v) fetal bovine serum (FBS) and 1% (v:v) of penicillin and streptomycin (all from GibcoBioCult, Paisley, Scotland, UK) used for the culture of HeLa and broblast cells. Incubation condition consisted of CO2 (5%) at 37 o C. Cell number and viabilities were de ned by hemocytometer and trypan blue staining. These cells were passaged via trypsin/EDTA (biosera, Franc).

MTT assay
The effects of nanocurcumin, curcumin and OA400 in HeLa and broblast cells viability were detected via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Brie y at rst the number of cells was measured by hemocytometer using trypan blue staining. Approximately 10 4 cells were seeded in each well of 96 well-plate, after incubation for 24 hours, cellular monolayers were formed, then treatment with nanocurcumin (10 μM -40μM), curcumin (10μM -70μM) and OA400 (10 μM -160 μM) were performed for 48 hours. Due to the fact that cancer cells are more sensitive to curcumin in compare with normal cells [18], broblast cells were treated with higher concentrations of this compound (shown in the results section). After the time required for treatment, the medium of each well was replaced with a solution containing MTT (0.5 mg ml −1 ) (Sigma Aldrich Company) and incubated for 4 h at 37 °C in 5% of CO2. Formazan was dissolved using 200μl of dimethyl sulfoxide (DMSO from Sigma Aldrich Company) and optical density (OD) of samples were measured at 540nm by plate Reader (BioTek Company, USA). Cell survival percentage was obtained by the absorption ratio of the treated cells to the control cells. The concentration at which 50% of the cells were destroyed considered as inhibitory concentration (IC50).

Cellular apoptosis analysis via owcytometry
In the apoptosis assay, HeLa and broblast cells were treated with curcumin (50μM) and nanocurcumin (15μM) according to IC50 value. After 48 hours cells were stained with Annexin V/ PI (propidium iodide) apoptosis detection kit according to the manufacturer's instruction (Ebioscience, Thermo Fisher Company). Apoptotic and necrosis levels of cells were detected via flow cytometry (FACS Calibur, USA).
RNA extraction, cDNA synthesis and real time RT-PCR Total RNA was extracted from treated and untreated HeLa and broblast cells with curcumin and nanocurcumin using TRIzol reagent (Invitrogen, Cat.no: 15596-026). Extraction steps were performed according to the instructions of Favorgen Biotech kit (Favorgen Biotech Company, Taiwan) Extracted RNAs were quali ed by UV spectrophotometry.
RNA was reverse transcribed using cDNA synthesis kit (Favorgen Biotech Company, Taiwan) according to its manufacture. DNaseI enzyme treatment was performed to ensure that RNA was not contaminated with DNA.
Quantitative measurement of E6, E7, P53 and Rb genes expressions was performed based on the comparative quanti cation method by real time RT-PCR in ABI Step One Sequence Detection System (Applied Bio-systems, CA, USA). In this method, SYBR® Premix Ex Taq™ II (TAKARA, Japan; Cat.RR82LR) was applied according to its manufacture and GAPDH gene utilized as an internal control. Speci c primers sequences of target genes have shown in table 1 and their concentrations were 10 pM in the reaction. 2 -ΔΔCt method was utilized to calculate he relative expression of genes. GraphPad Prism (version 7) software was determined the signi cance of the results.

Statistical analyses
Student t-test was used for deference examination between two groups. Statistical analyses were performed by GraphPad Prism 7 software. Probability values were set at the signi cant levels of p 0.05 (p 0.05 (*), p 0.01 (**) and p 0.001 (***).

Curcumin and nanocurcumin effect on HeLa and broblast cells viability
MTT calorimetric assay is a method that determine differences between viable cells and necrotic ones [19]. was about 15µM (Fig. 1A). This growth inhibitory effect of curcumin is much less than nanocurcumin. The value of IC50 for HeLa cells treated with curcumin for 48 hours has been increased to about 50µM (Fig. 1B). OA400 nanocarrier has no inhibitory effect on Hela cell growth. In this experiment, cells were treated with nanocarrier up to 160µM, but at none of the concentrations cell survival reached to50% (Fig. 1C).
Interestingly, nanocurcumin has not-signi cant effect on the growth of normal broblast cells. The IC50 value was observed at very high concentrations (about 160 µM, Fig. 2A). Pure curcumin and nanocarrier also had no signi cant inhibitory effect on broblast cell growth (Fig. 2B,C).

Nanocurcumin induce apoptosis in cancerous HeLa cells but not in normal broblast cells
Anxin-PI staining and ow cytometry was accomplished to evaluate the effect of nanocurcumin and curcumin on HeLa and broblast cells apoptosis. The results of owcytometry have shown that 46.5 percentages of HeLa cells that treated by 15µM nanocurcumin destructed through apoptosis process. Free curcumin with higher concentration (50µM) can induce apoptosis of 25.9% of cells. Thus, nanocurcumin is much more effective than curcumin (Fig. 3A). The apoptosis test for broblast cells has demonstrated that nanocurcumin (15 µM) and curcumin (50 µM) had no signi cant effect on the cellular apoptosis of these normal cells (Fig. 3B).
Nanocurcumin regulation effect on E6, E7, P53 and Rb mRNA expression The expression of E6 and E7 human papilloma virus genes and two related genes include P53 and Rb was measured in HeLa cells. Due to the absence of E6 and E7 oncogenes in broblasts, expression of P53 and Rb was measured in these cells under curcumin and nanocurcumin treatment. The results showed that in HeLa cells, E6 and E7 expression decrease after 48h of curcumin and nanocurcumin treatment, whereas P53 and Rb expression were enhance after this time in mRNA level with p-value < 0.05 (Fig. 4A,B). The alteration of these genes expression was more signi cant in nanocurcumin treatment condition. These ndings have demonstrated the positive regulation effect of nanocurcumin towards the apoptosis of cancer cells. In broblast cells that treated by nanocurcumin, the expression of p53 and Rb genes did not altered signi cantly (Fig. 4C). Therefore, nanocurcumin does not lead normal broblast cells to apoptosis process. This result is consistent with ndings from MTT and apoptosis assays.
Nanocurcumin regulates E7, P53 and pRb proteins level The expression of E7, P53 and phosphorylated Rb proteins were analyzed via western blot assay. The results showed that E7 protein level was reduced in nanocurcumin-treated Hela cancer cells after 48 hours (p-value < 0.05). The phosphorylated Rb and P53 proteins were increased in these cells signi cantly (p-value < 0.05) (Fig. 5). Western blot results con rm the ndings of the real-time RT-PCR assay.

Discussion
Cervical cancer is a common cancer in women. Many types of HPV virus infection cause about all cases of cervical cancer [5]. Due to the high prevalence of cervical cancer, drug resistance and systemic toxicity for conventional chemotherapy and radiation therapy, it is necessary to identify safe, effective and chemopreventive anti-cancer natural compounds. In this study, the positive regulation effects of nanocurcumin drug on the apoptosis and expression of E6 and E7 oncogenes and their associated tumor suppressor genes include P53 and pRb in human cervical cancer cell line HeLa cells were determined. HeLa cells are derived from the aggressive glandular cervical cancer of a woman and are used in many studies to evaluate cervical cancer treatment methods [20]. E6 and E7 oncogenes in the HeLa cell line derived from HPV genome. The integration of HPV type 16 or 18 DNA to HeLa cells genome has been proven in previous studies. HPV type 18 transcripts in the 8q24 chromosome region of HeLa cell line [21].
In the present study, it was found that nanocurcumin can increase cellular apoptosis and reduce cell viability in human cervical cancerous HeLa cells. Curcumin is a polyphenol compound derived from the root of the turmeric plant and has particularly function in combating cancer in many of studies. Curcumin is also well known as an anti-in ammatory and chemopreventative agent [22].
Studies in the last three decades about curcumin indicate poor bioavailability, hydrophobicity, poor cellular uptake and rapid metabolism of this component [23]. Various experiments have been performed to solve this problem, many of which have performed nanocarriers [24]. In the nanocurcumin that used for this study, OA400 nanocarrier resolved curcumin restrictions. It seems that the faster absorption of the drug is due to the increased permeability of the cell membrane by this nano-carrier.
Previous studies in Professor Sadeghizadeh's laboratory have developed nanocurcumin, including curcumin and OA400 nano carrier, and examined its anti-cancer properties in various cancers [17,25,26]. This nano-formulation of curcumin can affect different cellular pathways, including differentiation, cell proliferation and so on [12,27].
In this study, the effects of curcumin and nanocurcumin on HeLa cell viability and apoptosis were compared. It was found that nanocurcumin is much more effective than curcumin. This nding has been demonstrated in previous studies for other cancer cells [16] and may be due to the fact that nanocurcumin has higher cellular uptake and biostability than curcumin. HeLa and broblasts were treated separately with OA400 nanocarrier. The results have indicated that this carrier has no signi cant effect on cell apoptosis or cellular viability. The non-toxic effect of this nanocarrier proves its safety.
Nanocurcumin has progressed apoptosis process in Hela cancer cells in this experiment but has no signi cant effect on normal broblast cells in a dose dependent manner. This component at very high concentrations (160 µM) reduced broblast cell viability by approximately 50%. This concentration is not a functional concentration for studies. Therefore, it can be said that nanocurcumin is safe for normal cells at the concentration being studied. There are several reasons that explain why curcumin does not affect normal cells. For example, scientists have shown that the reduction of intracellular glutathione in cancer cells by butyonine sulfoxamine increases ROS levels and makes these cells more sensitive to curcumin [18]. Curcumin has also target molecules that are more common in cancer cells [28]. In previous studies, the capability of nanocurcumin to induce cancerous and undifferentiated cells apoptosis were detected in time and dose dependent manner, while on normal cells, no signi cant effects observed [29].
E6 and E7 play a key role in the pathogenesis of HPV. These genes encode proteins that bind to P53 and pRb tumor suppressors respectively and suppress them. This phenomenon disrupts the cell cycle regulation. Expression of these two viral oncogenes is essential for cervical cancer tumorigenesis [30].
In this study, the expression of E6 and E7 viral genes was signi cantly reduced in HeLa cells during treatment with nanocurcumin. The expression of two tumor suppressor factors affected by these genes, including P53 and Rb was also examined. As expected, following the expression decreasing of E6 and E7 genes, the expression level of P53 and Rb tumor suppressors increased both at the RNA and protein level. Thus, nanocurcumin provides a condition for the progression of apoptosis in Hela cancer cells and can be considered as a safe complementary drug for cervical cancer.
Curcumin has been applied in several other studies to apoptosis induction in cervical cancer, for example Zaman and his colleagues have evaluated another type of nanocurcumin (poly(lactic-co-glycolic acid) based curcumin nanoparticle formulation) to treat cervical cancer. In their results, as our ndings, nanocurcumin in 20µM and 25µM effectively inhibits the growth of cervical cancer cell lines, abrogating expression of E6 and E7 oncogenes, and leads them to apoptosis [15]. In another study investigators showed that curcumin at concentrations above 40 µM induced apoptosis in Hela cancer cells [31]. In present study nanocurcumin with lower concentration (15 µM) was functional. It con rms that nanocurcumin works more effectively and has more cellular uptake than curcumin.
The Expression of E6 and E7 oncogenes depends on the availability of the host transcription factors such as AP-1 family. Previous studies have shown that AP-1 activity be reduced via curcumin treatment. Curcumin also promote P53 expression by regulation of histone deacetylation and chromatin remodeling.
The induction of HeLa cell apoptosis by curcumin has been mediated by NFκB-P53-caspase3 pathway.