Sericin Preparation from Cocoons of Oak Tasar Silkworm Antheraea proylei J. Induces Apoptosis in a Caspase-dependent Manner in A549 and HeLa Cells and Caspase-independent Manner in PC3 Cells

estimated will be an protein of silk is a potential protein in various biomedical applications including cancer therapeutics. The present study evaluates the anticancer property of sericin prepared from cocoons of Antheraea proylei J. (A. proylei ) against human lung cancer (A549), cervical cancer (HeLa), and prostate cancer (PC3) cell lines. This is the rst report of the anti-cancer activity of the non-mulberry silkworm A. proylei. of the cell lines where A549 and HeLa cells are being non-malignant and p53 positive whereas PC3 cell is being malignant and p53 negative. The overall results of the present study envisage the possibility of using SP as an anti-tumorogenic agent.

cases of cancer in the year 2040 is 29.4 million per year globally (2). The increasing trend of cancer burden is due to several factors, including population growth, aging as well as the changing prevalence of certain causes of cancer linked to social and economic development particularly in rapidly growing economies. In contrast to other world regions, the proportions of cancer deaths in Asia and Africa (57.3% and 7.3%, respectively) are higher than the proportions of incident cases (48.4% and 5.8%, respectively), because these regions have a higher frequency of certain cancer types associated with poorer prognosis and higher mortality rates, in addition to limited access to timely diagnosis and treatment (2). According to the World Health Organization (WHO), the total number of cases of cancer diagnosed in India between the years 2017 and 2018 is 7,84,821 (increased by 324%) out of which 4,13,519 are men and 3,71,302 are women (1).
In view of the above facts, there is a need for urgent and serious attention towards nding new anticancer therapeutic drugs to prevent the increasing number of cancer cases in the coming decades in addition to other strategies. One such potential anti-cancer agent is sericin, a silk protein that binds together the silk broin bers to form the cocoon (3). Silk textile industry targets only the silk ber obtained after the process of sericin removal, through degumming (4). Depending upon the species from where the sericin is obtained, the amino acid composition varies considerably. Sericin from wild silkworms has a higher content of threonine, glutamic acid, cysteine and phenylalanine and a lower content of serine, proline, methionine, glucosamine, galactosamine, and histidine (5). Owing to the difference in the proportions of amino acid compositions in the domesticated silkworm, Bombyx .mori and the wild type silkworm, Antheraea sp., there may be variation in the bioactivity.
Sericin stands as a promising anti-cancer agent that inhibits the growth of cancer cells. The effect of sericin was studied in the colon cancer mice models induced by 1,2-Dimethlhydrazine (DMH). The studies concluded that sericin supplemented diet reduced the formation of colonic aberrant crypt foci (6). Further Zhaorigetu et al., (7) 2001 reported that sericin suppresses the development of colonic tumors by reducing oxidative stress, cell proliferation, and nitric oxide production. The strong antioxidant activity of sericin and its resistance to intestinal proteases prolongs its sustainability in the colon thereby lowering oxidative stress and tumorigenesis in the colon (7). Yet in another study, sericin was reported to suppress skin tumorigenesis in a mice cancer model induced by 7,12-dimethybenz (α) anthracene (DMBA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) by reducing oxidative stress, in ammatory responses and endogenous tumor promoter (TNF-α ) (8). It was also observed that sericin from B. mori induced apoptosis through the caspase pathway and downregulation of Bcl-2 expression in human colorectal cancer cells (SW480) (9). Most of the previous reports related to the prevention and treatment of colon cancer are concerned with sericin obtained from the commonly domesticated silkworm, Bombyx mori which has been extensively studied as compared to the wild silkworms including Antheraea sp. The oak Tasar silkworm, A. proylei is reared in several sericulture farms in Manipur, India and adjoining states and feeds on leaves of oak (Quercus sp.) which are naturally grown in the region. The sericin from A. proylei silkworm has yet to be explored for its prospective anti-cancer properties and health bene ts. Kumar et. al. 2019 (10) reported anticancer activities of sericin from non-mulberry silkworm Antheraea assamensis on MCF-7 and A431 cells through induction of oxidative stress and reduction of mitochondrial membrane potential. Further Zhang et al. 2003 (11), studied the potential of cecropins from Antheraea pernyi on inducing apoptosis in human colon adenocarcinoma cell lines. However, detailed studies including molecular mechanisms of inducing apoptosis have not been reported yet. Till now, the sericin from A. proylei has not been evaluated for its anticancer potential. The present study investigates the anticancer activity of the SP from the non-mulberry cocoon.

Methods:
Extraction of SP A. proylei cocoons were collected from Uyumpok Tasar Silk Farm, Imphal East, Manipur, India. Five grams of fresh cocoon cut (~ 1 cm 2 pieces) were added to 100 mL distilled water and subjected to heat treatment at 121°C under pressure for 1h. The resulting suspension was ltered through Whatmann lter paper No.1 and centrifuged at 21,000 g for 30 mins. The process was repeated for 2 times with the same cocoon shell sample. The supernatants obtained were pooled and then lyophilized. The lyophilized SP powder was stored at -20°C until use.
HPLC analysis of predominant amino acids of SP SP prepared as above was subjected to acid hydrolysis by dissolving in 6N HCl in boiling water bath for 24 h and mixed every hour for proper hydrolysis. It was then centrifuged at 42,000 g for 15 mins. The supernatant was ltered and neutralized with 1N NaOH. The ltered solution was then diluted to 1:1000 of the volume with milli-Q water and then analyzed for amino acids in HPLC (Agilent 1100 HP), C18, 4.5 X 150, 5µm column using mobile phase A (20 mM sodium acetate + 0.018% triethyalmine, pH to 7.20 ± 0.05) and mobile phase B (20% of 100 mM sodium acetate + 40% methanol and + 40% acetonitrile, pH 7.20 ± 0.05). The ow rate was maintained at 0.5mL/min and the column temperature was kept at 40C and detected at 338nm.
Cell treatment with SP SP was dissolved in RPMI culture media and centrifuged at 15,700g for 30 mins. The supernatant was sterilized through a syringe lter (0.2 µm pore size) for the treatment to the above three cancer cells with different doses ( nal concentration; 0. 2, 0.3, 1.0 , 1.5 , 3.0 , 5, and 10.0 µg/µL.) Cell viability assay Cell viability assay was carried out as per the manufacturer's protocol provided with MTT assay kit, Vybrant MTT assay Kit ( Invitrogen Life Technologies). A549, HeLa, and PC3 cells were cultured with a density of 1×10 4 cells per well in 100 µL RPMI (without phenol red), 10% FBS, and incubated with 5% CO 2 at 37 o C in a 96-well tissue culture plate. Various doses of SP were treated to the cells in triplicates. After 24 h, cell viability was assessed by adding 10μL of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL) to all the wells followed by incubation at 37 o C for 4 h. The formazan crystals formed were dissolved by adding 50µL of DMSO after removal of the culture media and further incubated for 10 mins at room temperature. The numbers of viable cells were then quanti ed by measuring absorbance at 540 nm. The experiment was conducted three times.

Comet assay
Comet assay was carried out according to the protocol described by Olive  About 400µl of the cell suspension was mixed with 1.2ml of low-melting agarose at 40 o C by gentle pipetting followed by pouring onto agarose pre-coated microscope slides. After proper solidi cation of the agarose, the slides were submerged in a neutral lysis solution containing 2% SDS, 0.5 mg/ml Proteinase K, 0.5M EDTA, and incubated at 37 o C for 16 h in the dark. The slides were then washed three times with a neutral rinse buffer followed by electrophoresis at 0.6 V/cm for 25 mins. Slides were then stained with 10µg/ml propidium iodide (PI) and observed under a uorescent microscope and photographed. The tail lengths of at least 100 comets in each slide were scored for analysis.
Western blots A549, HeLa, and PC3 cells were seeded in a 60 mm culture dish, grown overnight, and treated with various doses of SP. The cells were lysed using RIPA buffer and the protein concentration of the cell lysate was estimated using the BCA protein assay kit (Thermo Scienti c) and 20 µg of proteins were separated by12% SDS-PAGE. After transferring the separated proteins on PVDF membrane, it was blocked with 5% (w/v) skimmed milk in 1X Tris-buffered saline (TBS) with Tween-20 for 1 h at room temperature. The blocked membranes were then incubated with the primary antibodies (1:1000 dilution) with gentle shaking on a rocker at 4°C overnight. The membrane was probed with anti-PARP, anti-caspase 3 (both total and cleaved), anti-ERK, anti-P38 (both total and phosphorylated), or anti-JNK antibodies. The membranes were then washed with 1X Tris-buffered saline (TBS) with Tween-20 three times for 10 mins each with changes of buffer. After washing the membrane, secondary antibodies conjugated with horseradish peroxidase (HRP) were added at 1:1000 dilutions and kept with gentle shaking for 1h at room temperature. The membranes were re-blotted with an anti-ß actin antibody to normalize the total protein loaded. The antibodies were obtained from the Cell Signaling Technology, MA, USA. The blots were then developed using ECL (GE Amersham) and visualized under BioRad Gel Doc.
Cell cycle analysis A549, HeLa, and PC3 cells were cultured in a 6-well culture plate, grown overnight, and treated with various doses of SP. After 12 h, cells were harvested after trypsinization and washed with PBS. The cells were then xed with ice-cold 70% ethanol added dropwise with regular vortexing to avoid cell clumps and kept at 4°C for 30 mins. The xed cells were washed twice with PBS by centrifuging at 850 g for 5 mins.
Then the cells were treated with 50 µg of RNaseA (100µg/mL) and 200 µL of propidium iodide (50µg/mL) was added. Cell cycle distribution was analyzed in a FACS ow cytometer (BD Biosciences, USA).

Statistical Analysis
Signi cant variance between groups was performed for all groups using Student's t-test. Data are expressed as means ± SD. Differences with P <0.05 were considered statistically signi cant.

Results:
Amino acid composition of SP: The HPLC analysis of SP for constituent amino acids revealed the presence of aspartic acid, serine, glutamic acid, and histidine in the majority with other essential and non-essential amino acids. The free amino acid composition of sericin from Antheraea proylei and comparison with previous reports of sericin from Bombyx mori and Antheraea pernyi (12,13) is shown in Table  Table 1 Amino acid analysis of sericin preparation (SP) from Antheraea proylei by HPLC and comparison with previous reports of sericin from Bombyx mori and Antheraea pernyi The free amino acid composition of SP from Antheraea proylei was determined by acid hydrolysis with 6N HCl in boiling water bath and analysed using HPLC (Agilent 1100 HP). The amino acid content of sericin from Bombyx mori, and Antheraea pernyi is adapted from Yang et al., (12,13). The percentage of amino acid contents of sericin varies according to species. NA indicates Not Available SP induces cytotoxicity to A549, HeLa, and PC3 cells in a dose-dependent manner.

SP induces apoptosis through MAPK pathways
To determine the possible pathways of the apoptosis induced by SP, activation of MAPK pathways were assessed as it plays important roles in cell survival and death. Cells were treated with different doses of SP for 24 h and immunoblotted against the total as well as phosphorylated p38, p44, or SAPK/JNK antibodies. The results showed that SP leads to phosphorylation of p38, p44, and SAPK/JNK proteins in a dose-dependent manner in A549 and HeLa cells whereas in PC3 cells signi cant increase in phosphorylation in a dose-dependent manner is observed only in p38 protein. Phosphorylation of SAPK/JNK and ERK proteins in PC3 were observed only in the highest dose of SP [ Figure 4]. Therefore, SAPK/JNK and ERK pathway may not be directly involved in the SP induced apoptosis in PC3. The overall results indicate the involvement of p38, SAPK/JNK, and ERK pathways in A549 and HeLa cells whereas the p38 pathway but neither SAPK/JNK nor ERK pathways in PC3 are observed in the apoptosis induced by SP.
SP promotes cell cycle arrest at S phase in A549 and HeLa cells.
As seen with anticancer drugs, which arrests cell cycle at speci c points and thereby inducing apoptosis by destabilizing the normal biochemical processes of the cell, the anti-cancer activity of SP on A549, HeLa, and PC3 cells may affect the normal cell cycle. To determine whether the mechanism of action of SP for inducing apoptosis is due to the arrest of the cell cycle, cell cycle analysis was performed. Cells treated with varying doses of the SP for 12 h were subjected to ow cytometry analysis after staining with propidium iodide. An increase in the cell population at the S phase was observed in A549 as well as HeLa cells treated with SP. Although unpaired t-test between control and cells treated with 8µg/µL showed a statistically signi cant increase in cells at S phase in both HeLa and A549 cells (p<0.05), there was no statistically signi cant change in the population of cells at G0/G1 stage in both the cell lines. Reduction in the cell population at the G2/M phase was also observed in both the cell lines with a statistically signi cant difference observed between control and 8µg/µL. Contrastingly, cell cycle analysis of PC3 cells treated with SP showed cell arrest at the G1 phase in a statistically signi cant manner between control and treated cells (p<0.05), and also a consistent population was seen in S phase. We also observed a reduction of the number of cells at the G2M phase in a dose-dependent manner with statistical signi cance compared to untreated cells (p<0.00) [ Figure 5].

Discussion:
Sericin from B. mori has been reported to have anti-proliferative effects against colon cancer by downregulating the expression of Bcl-2, an anti-apoptotic protein, and activating the caspase-3 pathway (9). The chemopreventive effect of sericin in colon tumors was demonstrated by Zhaorigetu et al., 2007 (15) indicating that consumption of sericin reduces colonic oxidative stress and development of aberrant crypt foci due to its high content of serine (hydroxyl group) and the protease-resistant property which makes it readily absorbed by the large intestine and therefore can be used as a dietary supplement (15). However, the amino acid composition SP of A. proylei is different from those reported earlier in mulberry and non-mulberry silkworms [ Table 1]. This corroborates with earlier studies of sericin from different species of silkworms [5,16] The present study aims to evaluate the anti-cancer potential of SP on three types of cancers namely lung cancer, cervical cancer, and prostate cancer using A549, HeLa, and PC3 cell lines. Though such properties were observed in sericin of B. mori, it is the rst report on SP from the cocoon of A. proylei. Initially, dosedependent inhibition of cell proliferation of A549, PC3 and HeLa cell lines was observed as assessed by MTT assays and morphological changes of these cell lines were also observed under a simple microscope after SP treatments. Morphological changes of cells like rounding up of cells and bleb like structures which are the signs of possible programmed cell death were induced by SP [ Figure 1]. However, certain cell death pathways such as autophagy and apoptosis share similar morphological changes (17).
To negate the possibility of undergoing cell autophagy, expression of autophagy-related genes; ATG1, ATG-5, DRAM, and LC3 were assessed by semi-qPCR after SP treatments. No change in the expression of the autophagy-related genes was observed (data not shown). However, comet assay revealed that SP signi cantly induces genotoxicity in a dose-dependent manner suggesting induction of cell apoptosis [ Fig. 2]. Further molecular events leading to genotoxicity were investigated using Western blot analysis in which a caspase-3 and PARP dependent cell death was observed in A549 and HeLa cells whereas caspase-3 and PARP independent mechanism was observed in PC3 cells [ Figure 3]. The difference in the dependent of caspase-3 and PARP may be due to differences in the genotypes of the cancer cell lines where PC3 cells are highly malignant and p53 negative, whereas HeLa and A549 cells are non-malignant and p53 positive. Cancer cells have evaded normal cell death through a plethora of molecular changes and evading caspase-dependent cell death is one of the mechanisms observed in many cell lines. The nding in the present studies that SP induces PC3 apoptosis independent of caspase and PARP is parallel with the ndings of previous studies on plant extracts (18,19,20). Therefore, the ndings of caspase and PARP involvement in the case of A549 and HeLa cells but not in the case of PC3 cells suggest following different pathways for inducing cell apoptosis.
To investigate the signaling pathways of the apoptosis induced by SP, MAPK pathways were selected since it plays important roles in cell survival and cell death. The ndings in our study indicate that SP induces apoptosis in A549 and HeLa cells through activation of p38, SAPK/JNK, and ERK pathways.
However, SP induces cell apoptosis through p38 pathways activation but not SAPK/JNK and ERK pathways in the case of PC3 [ Figure 4]. Our observations are in agreement with earlier studies in which p38 and JNK are stress-activated involved in apoptosis of A549 cells (21,22), HeLa (11) and PC3 cells (23,24). Although the role of ERK in apoptosis remains controversial it is observed that DNA damage can induce ERK phosphorylation and further leading to cell death (25). Moreover, the role of ERK in cell death is dependent on cell lineage and intensity as well as the duration of pro-or anti-apoptotic signals of ERK1/2. Our observation of high-level phosphorylation in PC3 cells in the highest dose may be a result of extensive DNA damage activating ERK for its pro-apoptotic signal in a p53 independent manner (26).
Arrest of cell cycle at speci c points is a complex molecular mechanism and our observations of blockage of cell cycle progression induced by SP at G0/G1 in PC3 and S phase in A549 and HeLa reveal an interesting phenomenon suggesting different mechanisms of cell cycle arrests for different cell lineages [ Figure 5]. Further investigation is warranted to explain the molecular mechanisms of different cell cycle arrest induced by SP in prostate cancer.

Conclusion:
SP induces apoptosis in lung, cervical, and prostate cancer cell lines as observed in the assessment of cell death and genotoxicity with IC 50 values of 3.4 to 3.9 µg/µl through activation of MAPK pathways.
However, A549 and HeLa cells follow a molecular mechanism of caspase and PARP dependent while in PC3, it is a caspase and PARP independent mechanism. Further SP induces apoptosis in A549 and HeLa cells through activation of p38, SAPK/JNK, and ERK pathways but in PC3 SP induces cell apoptosis through p38 pathways activation. The difference in the molecular mechanisms of apoptosis induced by SP in A549, HeLa, and PC3 cell lines may be due to the difference in the genotypes of the cancer cell lines where A549 and HeLa cells are being non-malignant and p53 positive whereas PC3 cells are being highly malignant and p53 negative.
The overall results of the present study envisage the possibility of using SP as an anti-tumorogenic agent. The study is limited to the fact that the cellular protein that used for sericin binding needs to be determined. Further, the study opens up avenues for use of peptides that can act as anti-cancer agents which can increase the speci city and e cacy of drug designs in the future. The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.

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