Anti-tumour Effect of Cyanidin-3-o-glucoside Combined With Cisplatin in the Mice Xenograft Models of Cervical Cancer

Cervical cancer is the fourth most common carcinoma in women. Cisplatin (DDP) is the rst-line drug for the treatment of cervical cancer. Although ecacious, its application is constrained by the intolerance and serious adverse effects associated with cisplatin. Here, we aimed to investigate the in vivo anti-cervical cancer effects of cyanidin-3-o-glucoside (C3G), a type of anthocyanin, and DDP, when used alone or in combination; a BALB/c nude mouse xenograft tumour model was used. The tumour was inhibited in the three treatment groups when compared with untreated controls. The inhibition of tumour was 40.49%, 50.15%, and 58.49% when treated with C3G alone [40 mg/kg body weight (bw)], DDP alone (3 mg/kg bw), or a combination of C3G and DDP, respectively. Immunohistochemistry analysis indicated that treatment with C3G, DDP, or the combination induced apoptosis in xenograft tumours. Furthermore, after treatment, Bcl-2 level was decreased, Bax and cleaved caspase-3 expression was activated, and the PI3K/AKT/mTOR signalling pathway was modulated. These results suggest that the combination of C3G and DDP may have signicant synergistic anti-tumour ecacy in patients; therefore, this combination therapy has great potential for the treatment of cervical cancer.


Introduction
Cervical cancer is a malignant tumour in the female genital organ with high incidence worldwide, seriously affecting the quality of life and health of the patient 1 . It ranks high up on the lethality list among gynaecological malignant diseases 2 . Currently, surgery, radiotherapy, and chemotherapy are the main treatment methods for early-stage cervical cancer, which can signi cantly prolong and improve the survival time and quality of life of most patients 3 . Chemotherapy is an important clinical treatment option for cancers, and cisplatin (DDP) is one of the most effective anti-cancer drugs to treat metastatic cervical cancer 4,5 . However, intolerance to it and adverse effects has limited its use in the clinic 6,7 . Therefore, there is still an urgent need for the development of novel effective therapeutic agents for the treatment of this aggressive malignant cancer.
In recent years, the close relationship between human health and diet has prompted us to explore bioactive chemicals in fruits and vegetables. Polyphenols, including anthocyanins, phenolic acids, and avonoids, are the most important compounds with health bene ts in fruits and vegetables 8 . Previous studies have shown that anthocyanins have a wide range of biological activities, including anti-oxidant, anti-cancer and anti-ageing [9][10][11] . Polyphenols interfere with carcinogenesis by targeting multiple signalling pathways and inducing apoptosis, while sparing the survival pathways in normal cells. Polyphenols are also be used as adjuvants to enhance the overall e cacy of conventional chemotherapy drugs 13 .
There is in vivo evidence that non-nutritional C3G exhibits numerous health-promoting effects including anti-oxidant, cardioprotective, and anti-cancer properties [14][15][16] . However, the potential of C3G as a possible co-adjuvant of DDP in cervical cancer therapy is yet to be evaluated.
The phosphatidyl inositol 3-kinase (PI3K/AKT/mTOR) signalling pathway is involved in the regulation of cell proliferation, migration, and apoptosis 17 . Numerous studies have shown that the inhibition of expression of phosphatidylinositol 3-kinase (PI3K) protein may play an important role in impeding tumour growth 18,19 , whereas the activation of the PI3K/AKT/mTOR pathway may be related to the serious side effects of chemotherapeutic drugs 20 . Previous studies have shown that the DDP-induced nephrotoxicity reduced by inhibiting the PI3K/AKT/mTOR pathway 21 . Therefore, it could be hypothesised that C3G combined with DDP might play a therapeutic role by regulating PI3K/AKT/mTOR pathway.
Previous studies have shown that the C3G exerted anti-tumour effects, but to the best of our knowledge, this the rst time that an in vivo study combing C3G and DDP has been tested in a cervical cancer mouse xenograft model.

Results
The effect of C3G, DDP, or their combination on the body weight of nude mice The change in body weight is the fundamental method to detect acute toxicity of any test substance.
Generally, the acute toxicity of a drug is evaluated by examining whether the body weight of the mice decreases sharply after 72 h of treatment 22 . We proceeded to investigate the potential therapeutic bene ts of C3G with DDP in vivo using mice xenograft models.
Tumors were allowed to grow for one week, after which mice were treated with C3G, DDP, or C3G+DDP for another 2 weeks (Fig. 1A). At the end of the study, we observed that DDP caused a reduction in the animal's body weight when compared with the model group (P < 0.05; Fig. 1B). There was no signi cant change in body weight in mice when we compared the C3G or C3G+DDP group, with the model group (P > 0.05). In addition, the mice in the C3G and the C3G+DDP groups were more lively and active than the mice in the DDP group. These results indicate that anti-cancer drug (DDP) affected the body weight in mice with tumours and that C3G alleviated, to some extent, the weight loss in tumour bearing mice.
The effect of C3G, DDP, or their combination on tumour weight and tumour growth At the end of the experimental period, tumour tissues were excised and their volumes and weighst were measured. As shown in Figures 2A and 2B, the tumour growth was suppressed in all the treatment groups, relative to that in the model group. The tumour volumes of the combination group were signi cantly smaller than in the model group (P < 0.01), which indicates that the inhibitory effects on tumour volumes were much stronger by the combination treatment of C3G and DDP.
Tumour weight in all treatment groups decreased signi cantly relative to those in the model group on day 22 (P < 0.01; Fig. 2C). A combination of C3G (40 mg/kg/d) and DDP 3 mg/mg once in 3 days) decreased the tumour weights form 610 ± 56.25 mg in the model group to 253.2 ± 49.51 mg (P < 0.01) in the treatment group. As shown in Figure 2D, compared with the model group, the tumour growth was inhibited by 40.49%, 50.15%, and 58.49%, in the C3G, DDP and C3G+DDP groups, respectively. Thus, the combination of C3G and DDP inhibited tumour growth 8.34% more than DDP alone. In summary, the combination treatment could signi cantly reduce tumour weight and growth, suggesting that the anticancer effect of the combined treatment was signi cantly stronger than that of DDP alone.
The effect of C3G, DDP, or their combination on cell apoptosis of the xenograft tumour The persistence of tumour cells in the xenograft indicates that they have evaded the host immune system; therefore, an extraneous treatment is required to control uncontrolled proliferation 23 . We analysed the induction of apoptosis using the TUNEL assay, wherein fragmented DNA, a characteristic of apoptotic cells, is used to identify apoptotic cells. Cells uorescing red owing to fragmented DNA indicates apoptotic cells. Apoptosis was observed in all treated groups, compared with that of the model group. As shown in Figure 3A, the area of uorescence in the xenograft tumours signi cantly increased in mice treated with the combination of drugs (C3G + DDP) compared with that in mice treated with either C3G or DDP alone. Figure 3B shows that the percentage of cells undergoing apoptosis increased upon treatment with either C3G or DDP alone compared with the model. The percentage of apoptotic cells in the tumours of mice treated with the combination of drugs (C3G + DDP) was 15.85% more than in the tumours of mice treated with either drug alone.
The effect of C3G, DDP, or their combination on apoptotic proteins in xenograft tumour To demonstrate the pro-apoptotic effects of C3G alone or in combination with DDP on xenograft tumour, we analysed the expression levels of Bax, Bcl-2, and cleaved caspase-3, known as apoptosis regulators. As shown in Figure 4, the xenograft tumour cells were stained with DAB that could speci cally bind to Bax, Bcl-2, and cleaved caspase-3. The related proteins were imaged as blue uorescent areas. The results indicate that the percentage of Bcl-2 protein-positive cells signi cantly decreased in all treatment groups compared to that of the model (P < 0.05; Fig. 4C and 4D). In contrast, the percentage of Bax were signi cantly increased by 3.77%, 3.47%, and 7.23% and those of cleaved caspase-3 by 3.03%, 2.83%, and 4.92% in the C3G, DDP and the C3G+DDP groups, respectively, compared with model group (all P < 0.05; Fig. 4A, 4B, 4E, and 4F).
The effect of C3G, DDP, or their combination on the phosphatidyl inositol 3-kinase PI3K/AKT/mTOR pathway in the xenograft tumour The PI3K/AKT/mTOR pathway is involved in the regulation of cell proliferation and apoptosis [24][25][26] . Activation of the PI3K/AKT/mTOR signalling pathway is critical for tumour cell growth and survival in several solid cancers 27 . Therefore, to understand the molecular processes underlying the effects of C3G and DDP on xenograft tumour, we examined the expression of the signalling proteins involved in the PI3K/AKT/mTOR pathway.
The results indicate that the percentage of p-AKT, p-PI3K, and p-mTOR protein-positive cells were less in all treatment groups compared with the C3G+DDP group; less by 4.57%, 6.04%, and 6.16%, in the C3G, DDP, and C3G+DDP groups, respectively (Fig. 5). Furthermore, the percentage of these signalling-proteinspositive cells may explain why the combinational treatment exhibited stronger apoptotic effects in tumour tissues than those exhibited by the treatment with C3G or DDP alone. This study suggests that the observed apoptosis in the xenograft tumour apoptosis caused by DDP or C3G is regulated by t p-AKT, p-PI3K, and p-mTOR.

Discussion
Cervical cancer is among the most common malignant disease for women, ranking the second in the incidence list of gynaecological cancer. DDP, a classical rst-generation platinum drug, is widely used as a therapy for cervical cancer. DDP resistance is related to a variety of mechanisms, such as DNA repair, increased drug inactivation (preventing drug forms from reaching their DNA targets), growth signalling pathways, and increased expression of anti-apoptotic proteins 28,29 . The application of DDP is limited owing to its adverse effects and tumours developing drug resistance 30,31 .
The therapeutic index, which takes into account the toxicity of a durg at its e cacious dose, is an important factor considered while developing chemotherapy drugs. Numerous studies have looked at combining various monotherapy drugs to combat various diseases, including cancer, to improve their therapeutic index 32 . In this study, we found that C3G could enhance the e cacy of DDP by reducing the tumour weight and volume, without any observable toxicity in mice at the tested dose. These results demonstrate that C3G can improve the therapeutic effect of DDP, thereby reducing the adverse effects of DDP.
Furthermore, we studied the underlying mechanisms by which the C3G+DDP combination inhibits xenograft tumour cell growth. Apoptosis is an active process of cell death, characterised by cell contraction, chromatin aggregation with genome fragmentation, and nuclear pyknosis 33 . Promoting cancer cell apoptosis is a key characteristic of chemotherapy drugs 34 . Mitochondria are highly dynamic organelles in eukaryotic cells and play an important role in the process of apoptosis 35 . It is widely known that the Bcl-2 family of proteins play an important role in cell apoptosis and includes the pro-apoptotic Bax protein and the anti-apoptotic Bcl-2 protein 36 . Several studies have established that the downregulated and upregulated expression of Bcl-2 and Bax, respectively, could induce mitochondrial outer membrane permeability, resulting in the release of a variety of apoptotic proteins and cell death mediators 37,38 . Caspase-3 is the main effector of programmed cell death and is known to initiate most of the apoptotic signalling cascades 39 . We found that the C3G+DDP combination could signi cantly induce cell apoptosis, compared with C3G or DDP alone. These ndings suggest that the C3G+DDP combination has a synergistic effect as demonstrated by the increased percentage of cells expressing Bax and cleaved caspase-3 in xenograft tumorus, thereby inducing an elevated level of xenograft tumour cell apoptosis.
Numerous studies have demonstrated that the PI3K/AKT/mTOR pathway plays a central role in the growth, survival, and motility of cancer cells, making it an important target for anti-tumour drug development 40 . Inhibition of signalling along this pathway could lead to increased cell death 41 . To examine the effect of C3G on this pathway, we analysed key proteins of the PI3K/AKT/mTOR pathway in mouse cervical tumour tissue. The imbalance in the expression of PI3K/AKT/mTOR signalling proteins affects the downstream mechanisms related to tumor development. AKT, also known as protein kinase B, is a key signalling effector of PI3K and an oncogene product that regulates cell growth 42 . Upon phosphorylation-dependent activation, AKT phosphorylates its downstream substrate mTOR, thus promoting cell growth and proliferation 43 , whereas inhibition of AKT phosphorylation can induce apoptosis 44 . Here, we found that the C3G+DDP combination signi cantly inhibited p-P13K, p-AKT, and p-mTOR expression, compared to other treatment groups.

Conclusion
Our previous studies had revealed that the combination of C3G and DDP could inhibit the growth of cervical cancer cells in culture. This study, to the best of our knowledge, is the rst to investigate the effects of the combination of C3G and DDP on cervical cancer cell-induced tumour growth in vivo. Application of DDP together with C3G reduced tumour weight, induced apoptosis in the xenograft tumours, and down-regulated PI3K/AKT/mTOR signalling. Our data provide evidence that DDP in combination with C3G may have therapeutic potential for the treatment of cervical cancer.

Materials And Methods
Chemicals and antibodies C3G (purity > 98%) was provided by Nanjing Plant Origin Biological Technology Co., Ltd. (Nanjing, China). DDP was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Antibodies were purchased from Wanlei Biological Technology Co., Ltd. (Shenyang, China); detailed information on each antibody is listed in Supplementary Table S1.

Cell culture
The human cervical cancer cell line (HeLa) was obtained from Wanlei Biological Technology and cultured in Dulbecco's Modi ed Eagle's Medium supplemented with 10% foetal bovine serum (FBS). Cells were maintained at 37°C in an incubator with a humidi ed atmosphere and 5% CO 2 .

Animal origin
Twenty-four BALB/c nude mice, 5-6 weeks-old, and weighing 18 ± 2 g were provided by Beijing HFK Bioscience Co. Ltd. The mice were raised at 25 ± 2°C, with free access to feed and water. All procedures and protocols of experiments were endorsed by the Ethical Committee for the Experimental Use of Animals at Shenyang Agricultural University (approval number 202011014) under the regulations of the "Regulations for the Administration of Affairs concerning Experimental Animals" guidelines and with the ARRIVE guidelines.

Animal model and treatments
The mice were fed for 5 days and fasted for 12 h before starting the xenograft study. HeLa cells(5 × 10 6 ) were subcutaneously injected into the right axilla to establish the mice cervical cancer model. After 24 h of tumour cell injection, mice were randomly divided into 4 groups with 6 mice per group: model group, C3G group, DDP group, and C3G+DDP group. One week after cell injection: model group, was administered gavage with 0.2 mL normal saline every day for 2 consecutive weeks; the C3G group was administered gavage with C3G [40 mg/kg body weight (bw), dissolved in distilled water] every day for 2 weeks; the DDP group, received intraperitoneal injections of DDP (3 mg/kg bw) once in three days for 2 weeks; and the C3G+DDP group was administered gavage with C3G (40 mg/kg bw) every day combined with intraperitoneal injection of DDP (3 mg/kg bw) once in three days for 2 weeks.

Measurement of tumour diameter, volume and tumour weight
After tumor formation, the diameter of the tumour growth was measured every 3 days and the volume of the tumour was calculated. Formula for tumour volume was as follows: tumor volume (mm 3 ) = [length diameter (mm) × short diameter (mm) × short diameter (mm)] / 2. At the end of 2 weeks of treatment, intraperitoneal infusion was performed using 10% chloral hydrate (3.5 mL/kg bw) to anaesthetise the mice, and tumours were removed for measurement of volume (mm 3 ) and weight (mg).
Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) test TUNEL staining was performed to detect cell apoptosis in tumour tissues. The analysis was conducted as previously described 45 . Tissues were xed in 10% formaldehyde and then embedded in para n blocks for sequential sectioning into 4-μm-thick sections. The tissues were washed under tap water for 4 h, subjected to a gradient ethanol series (70%, 80%, 90%, 100%, and 100%) for 1 h each, and then washed twice in PBS. Next, the tissues were treated with Proteinase K for 20 min at 37°C and washed with PBS. After drying, a 50 μL TUNEL reaction mixture was added, a cover glass was added on the slide, and the side was kept in a dark humid box at 37°C for 1 h. For the negative control group, only 50 μL uoresceinlabelled dUTP solution was added. The slides were washed thrice in PBS. After staining with DAPI for 10 min, the slides were washed thrice in PBS. A drop of PBS was added, and images were captured under the eld of vision with a uorescence microscope (BX53, Olumpus, Japan). The rate of apoptosis was calculated by determining the ratio of apoptotic cells to total cells using the Image-pro Plus 6.0 software. Immunohistochemisitry Immunohistochemistry was conducted as described previously 45 . Immunostaining was performed to observe and image the expression levels of Bax, Bcl-2, cleaved caspase-3, p-AKT, p-PI3K, and p-mTOR in the tumours. Brief, the tumour samples were dewaxed and hydrated at room temperature, and the endogenous enzyme activity was inactivated and heat-antigen-repaired. The rack was replaced with a new one, the slices were hydrated, and a circle was drawn using an immunohistochemical pen. Nonspeci c sites were blocked, the sections were incubated with primary antibodies and washed with PBS, and then incubated with the HRP-conjugated secondary antibody (Thermo Fisher Scienti c, Waltham, MA, USA). After washing with PBS and distilled water thrice successively, the sections were stained with 3, 3diaminobenzidine (DAB) and counterstained for 3 min with haematoxylin solution. The samples were differentiated with 1% hydrochloric acid alcohol and washed with water for 20 min, dehydrated, and mounted with neutral resin. Finally, the images were analysed using Image-pro Plus 6.0 software.

Statistical analysis
The data are presented as the mean ± standard deviation (SD). The differences between samples were analysed by using one-way analysis of variance (ANOVA) using Turkey's-b comparison and considered signi cant at P < 0.05. All calculations were performed using SPSS v. 26.0 (SPSS Inc., Chicago, IL).