Antheraea proylei J. sericin induces apoptosis in a caspase dependent manner in A549 and HeLa cells and caspase independent manner in PC3 cells

Background In spite of advancement early the expanding of and the global burden is still signicant and It is estimated that the new cases of cancer in the year 2040 will be 29.4 million per year globally. Sericin, an adhesive protein of silk cocoons has been shown to be a potential protein in various biomedical applications including cancer therapeutics. The present study evaluates the anticancer property of sericin from cocoons of Antheraea proylei J. (SAP) against human lung cancer (A549), cervical cancer (HeLa), and prostate cancer (PC3) cell lines. This is the rst report of anti-cancer activity of the non-mulberry silkworm A. proylei J. cell lines. However, further investigation is warranted. The overall results of the present study envisage the possibility of using SAP as anti-tumorigenic agent.


Introduction
Although we have made much progress in understanding the biology of cancer disease, advancement in technology for early diagnosis, the expanding array of anticancer drugs, and treatment modalities, the global cancer burden remains signi cant and increasing. It is estimated that new cases of cancer have risen to 18.1 million and deaths due to the disease to 9.6 million in 2018 [1]. Sadly, the estimated new cases of cancer in the year 2040 is 29.4 million per year globally. 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 [2,3]. In contrast to other world regions, the proportions of cancer deaths in Asia and in 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. The total number of cases of cancer diagnosed in India in between the years 2017 and 2018 is 7,84,821 (an 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 which binds together the silk broin bres to form the cocoon [4]. Silk textile industry targets only the silk bre obtained after the process of sericin removal, through degumming [5].
Sericin stands as a promising anti-cancer agent which 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 [6,7].
Further Zhaorigetu et al., (2001) reported that sericin suppresses the development of colonic tumours by reducing oxidative stress, cell proliferation, and nitric oxide production [8]. 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. Yet in another study, sericin was reported to suppress skin tumorigenesis in mice induced by 7,12-dimethybenz (α) anthracene (DMBA) and 12-Otetradecanoylphorbol 13-acetate (TPA) by reducing oxidative stress, in ammatory responses and endogenous tumour promoter (TNF-α ) [9]. It was also observed that sericin from B. mori induced apoptosis through caspase pathway and downregulation of Bcl-2 expression in human colorectal cancer cells (SW480) [10]. Kumar & Mandal, (2019) [11] reported anticancer activities of sericin from nonmulberry 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) [12], 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 is poorly understood.
The sericin used in most of the previous studies on the prevention and treatment of cancer were obtained from Bombyx mori or Antheraea sp. which have been extensively studied.
Depending upon the species of silkworm, the amino acid composition of sericin varies considerably.
Sericin from wild silkworms have a higher content of threonine, glutamic acid, cysteine and phenylalanine and a lower content of serine, proline, methionine, glucosamine, galactosamine and histidine [13]. Therefore, sericin from different species of silkworm may have different biological activity and e cacy. The oak tasar silkworm, A. proylei J. is reared in several sericulture farms in Manipur and adjoining states and feeds on leaves of oak (Quercus sp.) which are naturally grown in the region. The sericin from A. proylei J. silkworm has yet to be explored for its prospective anti-cancer properties and health bene ts. Therefore, the present study aims to evaluate the anticancer property of sericin prepared from cocoons of Antheraea proylei J. (SAP) against human lung cancer (A549), cervical cancer (HeLa), and prostate cancer (PC3) cell lines. This is the rst study of anti-cancer activity of the non-mulberry silkworm A. proylei J. to the best of our knowledge.

Methods:
Extraction of SAP A. proylei J. cocoons were collected from Uyumpok Tasar Silk Farm, Imphal East, Manipur (24 o 57' 2.7396" N, 94 o 2' 55.2282" E), 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 1hr. The resulting suspension was ltered through Whatmann lter paper No.1 and centrifuged at 21,000g 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 SAP powder was stored at -20°C until use.
HPLC analysis of predominant amino acids of SAP: SAP was subjected to acid hydrolysis by dissolving in 6N HCl in boiling water bath for 24 hrs and mixed every hour for proper hydrolysis. It was then centrifuged at 3500 rpm 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. After 24 hrs, cell viability was assessed by adding 10μL of 3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) to all the wells followed by incubation at 37 o C for 4 hrs. 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 570 nm. The experiment was conducted in triplicates at least three times.

Comet assay
Comet assay was carried out according to the protocol described by Olive and Banath, 2006 [14]. Western blots A549, HeLa or PC3 cells were seeded in a 60 mm culture dish, grown overnight, and treated with various doses of SAP. 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 with 0.1% Tween-20 (TBST) for 1 hr 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 antibodies anti-PARP, anti-caspase 3 (both total and cleaved), anti-ERK, anti-p38 or anti-JNK antibodies (both total and phosphorylated) (Cell Signalling, USA). The membranes were then washed with 1X TBST 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 1hr at room temperature. The membranes were re-blotted with an anti-ß actin antibody to normalize the total protein loaded. The blot was then developed using ECL (GE Amersham) and visualized under BioRad Gel Doc.

Results:
Amino acid composition of SAP: The HPLC analysis of SAP 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 [ Table 1]. The free amino acid composition of SAP from Antheraea proylei and comparison with previous reports of sericin from Bombyx mori and Antheraea pernyi [15,16] is shown in Table 1 SAP induces cytotoxicity to A549, HeLa or PC3 cells in a dose dependent manner.

SAP induces genotoxicity
To ascertain the cytotoxic activity induced by SAP on A549, HeLa and PC3 cells is due to induction of apoptosis, genomic DNA fragmentation was assessed by comet assay. Cells were treated with different doses of SAP (control, 2µg/µL, 4µg/µL and 8µg/µL) for 24 hrs and comet assays were performed as described above.

SAP induces A549 and HeLa cells apoptosis through caspase-3 activation and PARP deactivation while in PC3 independent of caspase and PARP
To determine the molecular mechanism of the apoptosis induced by SAP, the activation of executioner caspase-3 and deactivation of PARP in A459, PC3 and HeLa cells were assessed by Western blotting. The three cell lines were treated with various doses of SAP for 24 hrs and immunoblotted against caspase-3 or PARP antibodies. The results showed that treatment of SAP to A549 and HeLa cells leads to the cleavage of caspase-3 and PARP proteins. On the other hand, in PC3 cells no cleavage of caspase-3 and PARP proteins was observed [ Figure 3]. When assessed the cleavage of caspase-7 and caspase-9, cleavage of none was observed in case of PC3 (result not shown). The results suggest that SAP induces cell apoptosis in a caspase-and PARP-dependent manner in A549 and HeLa cells but in a caspase-and PARP-independent manner in PC3 cells.

SAP induces apoptosis through MAPK pathways
To determine the possible pathways of the apoptosis induced by SAP, activation of MAPK pathways was assessed as it plays important roles in cell survival and death. Cells were treated with different doses of SAP for 24 hrs and immunoblotted against the total as well as phosphorylated p38, ERK, or SAPK/JNK antibodies. The results showed that SAP leads to phosphorylation of p38, ERK, 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 SAP. Therefore, SAPK/JNK and ERK pathway may not be directly involved in the SAP induced apoptosis in PC3 [ Figure 4]. To con rm the involvement of ERK, p38 or SAPK/JNK in apoptosis of HeLa and A549 cells, speci c inhibitors for ERK, p38, and SAPK/JNK were used and the results revealed role of p38 in HeLa and ERK in A549 cells [ Figure 5] as the pre-treatment of SB203580 and FR180204 could rescue the A549 and HeLa cell death from SAP treatment respectively.
SAP 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 SAP on A549, HeLa and PC3 cells would have affected the normal cell cycle. To determine whether the mechanism of action of SAP for inducing apoptosis is due to the arrest of the cell cycle, cell cycle analysis was performed. Cells treated with varying doses of the SAP for 12 hrs were subjected to ow cytometry analysis after staining with PI. An increase in the cell population at the S phase was observed in A549 and HeLa cells treated with SAP. Although independent t-test between control and cells treated with 8µg/ µL (2xIC 50 ) 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 between control and cells treated with 8µg/µL. Contrastingly, cell cycle analysis of PC3 cells treated with SAP showed 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 [ Figure 6]. 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<0.01).

Discussion:
Sericin protein is an important bioresource with many biological applications that is underutilized and segregated as waste by silk industries. Earlier studies on sericin from B. mori have indicated pharmaceutical and cosmetic applications as well as anti-cancer properties on cancer cell types and mouse models pointing towards use of the protein as nutritional supplements in preventive medicine. Moreover, recent advancement in use of peptides as anti-cancer agents further opens up avenues for use of naturally available peptides for the purpose. With difference in species of the silk moth and diverse feeding habits, the composition and texture of silk bres are different owing to variation in economic values. Amino acid composition analysis of sericin from B. mori and A. pernyi revealed that the two sericin have different amino acid composition [13,[15][16][17]. Therefore, amino acid composition of sericin from Antheraea proylei J. was analysed and the results revealed that amino acid composition of sericin of Antheraea proylei J. is different from that of B. mori and A. pernyi [ Table 1]. Our ndings suggest that sericin protein is highly diverse and may have different bioactivities. Analysis of cell cytotoxicity and anticancer evaluations remains limited to domesticated species of silkworm, B. mori. Our nding is the rst of its kind report on the anti-cancer property of sericin from A. proylei J. cocoons.
Our analysis of sericin from A. proylei J. on cell cytotoxicity by microscopy indicated signs of abnormal cell morphology such as detachment, round up structures and signs of membrane blebbing. We further determined the IC 50 value of SAP on the three cell lines by MTT assay which indicated a dose-dependent response on all the three cell lines with different IC 50 values [ Figure 1(g)]. The difference in IC 50 values of the three cell lines may be due to difference in susceptibility of sericin as observed in various earlier studies with other agonists [18,19]. Our study is in agreement with earlier ndings of sericin from B. mori that SAP has anti-proliferative activity [10].
Certain cell death pathways such as autophagy and apoptosis share similar morphological changes [20].
To negate out the possibility of undergoing cell autophagy, expression of autophagy related genes; ATG1, ATG-5, DRAM and LC3 were assessed by semi qPCR after SAP treatments. No change in the expression of the autophagy related genes was observed (data not shown). However, comet assay revealed that SAP signi cantly induces genomic DNA fragmentation in a dose-dependent manner strongly suggesting induction of cell death through apoptosis [ Figure 2].
Further molecular events leading to genomic DNA fragmentation 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 as well as PARP independent mechanism was observed in PC3 cells. The difference on the dependence of caspase-3 and PARP may be due to difference in the genotypes of the cancer cell lines or the type of agonists used. 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 SAP induces PC3 apoptosis independent of caspase and PARP is parallel with the ndings of previous studies on plant extracts [21][22][23]. Caspase-3, a member of the caspase family plays a central role in inactivation of PARP by cleavage at DEVD site leading to fragmentation of the chromosome to 50kb fragments [24]. In case of HeLa and A549, our observation of caspase-3 activation and PARP deactivation after subsequent exposure to SAP corroborates the fragmented DNA observed in comet assay. Interestingly, absence of caspase-3 activation and PARP inactivation in PC3 warrants a different mechanism leading to DNA fragmentation and apoptosis needing further analysis of the mechanism.
To investigate the signalling pathways of the apoptosis induced by SAP, MAPK pathways were selected since it plays important roles in cell survival and cell death. The ndings in our study indicate that SAP induces apoptosis in A549 and HeLa cells through activation of p38, SAPK/JNK and ERK pathways. Inhibition of p38 rescued cells from apoptosis in HeLa cells whereas inhibition of ERK phosphorylation had similar consequence in A549 cells. The ndings indicate role of p38 and ERK in trigger of apoptotic pathway in HeLa and A549 cells induced by SAP [ Figure 7]. However, SAP induces cell apoptosis through p38 pathways activation but not SAPK/JNK and ERK pathways and independence of caspase-3 and PARP in the case of PC3. Our observations are in agreement with earlier studies in which p38 and JNK are reported as stress activated and involved in apoptosis of A549 cells [25], HeLa [26] and PC3 cells [27,28] 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 [29]. Moreover, the role of ERK in cell death is dependent on cell lineage and intensity as well as duration of pro-or anti-apoptotic signal of ERK1/2. Our observation of high intensity phosphorylation in PC3 cells in the highest dose may be a result of extensive DNA damage activating ERK for its pro-apoptotic signal [30].
Arrest of cell cycle at speci c points is a complex molecular mechanism and our observation of blockage of cell cycle progression induced by SAP at G0/G1 in PC3 and S phase in A549 and HeLa reveals an interesting phenomenon suggesting different mechanism of cell cycle arrests for different cell lineages. Further investigation is warranted to explain the molecular mechanisms of different cell cycle arrest induced by SAP.

Conclusion:
The SAP in this study induces apoptosis in lung, cervical, and prostate cancer cell lines as observed in the assessment of cell death and genomic DNA fragmentation 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 caspaseand PARP-dependent while in PC3 it is a caspase-and PARP-independent mechanism. Further, SAP induces apoptosis in A549 cells through activation of ERK and in PC3 and HeLa cells through p38. The difference in the molecular mechanisms of apoptosis induced by SAP in A549 and HeLa cell lines and in PC3 cell lines may be due to the difference in the genotypes of the cancer cell lines.
The overall results of the present study envisage the possibility of using SAP as an anti-tumorigenic agent. The study is limited to the fact that the target cellular proteins where SAP binds 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.   Schematic presentation of molecular pathways of apoptosis induced by SAP: A549 and HeLa cells treated with inhibitor of ERK and p38 phosphorylation respectively could rescue the cells from apoptosis induced by SAP indicating different mechanisms of cell death in different cell lines.