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ORIGINAL ARTICLE
Year : 2021  |  Volume : 17  |  Issue : 5  |  Page : 96-104  

Apoptosis-inducing and antiangiogenic activity of partially purified protein from the pericarp of Zanthoxylum rhetsa in vitro and in vivo


1 Post-Graduation Department of Biotechnology, Teresian College, Teresian Research Center (Affiliated to the University of Mysore), Mysuru, Karnataka, India
2 Department of Biochemistry, Chika Aluvara PG Center, Mangalore University, Mangalore, Karnataka, India
3 Post-Graduation Department of Biotechnology, Teresian College, Teresian Research Center (Affiliated to the University of Mysore), Mysuru; Department of Studies and Research in Food Technology, Davengere University, Davengere, Karnataka, India

Date of Submission04-Dec-2020
Date of Decision23-Feb-2021
Date of Acceptance27-Mar-2021
Date of Web Publication10-Jun-2021

Correspondence Address:
Shankar Jayarama
Post-Graduation Department of Biotechnology, Teresian Research Foundation (Affiliated to the University of Mysore), Siddhartha Nagar, Mysuru - 570 011
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_520_20

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   Abstract 


Background: Biological activities of Zanthoxylum rhetsa have been well studied, and its various parts have been reported to possess anticancer activities. The reports on anticancer activities of proteins from the fruits of Z. rhetsa are limited. Objectives: The study assessed the proapoptotic and antiangiogenic activity of partially purified proteins from the pericarp of Z. rhetsa. Methods: MCF-7, MDA MB 231, HeLa, and HCT 116 cells treated with partially purified protein fractions of Z. rhetsa pericarp were assessed for cytotoxicity by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-diphenyl tetrazolium bromide (MTT) assay. Fraction ZR3 showed high cytotoxicity against the MCF-7 cells; hence, it was chosen for further assays. The proapoptotic activity was scrutinized by Giemsa staining, acridine orange–ethidium bromide staining, and DNA fragmentation assay. The study was supported by wound healing assay and colony formation assay. Cell cycle analysis was performed by fluorescence-activated cell sorter. Ehrlich ascitic carcinoma-bearing Swiss albino mice were used as in vivo model. Angiogenesis was studied by peritoneal angiogenesis in mice and chorioallantoic membrane assay in fertilized eggs. Results: MTT assay showed the inhibition of MCF-7 cells (IC50 = 21.5 μg/mL) by ZR3 fraction. Reduction in proliferation and failure to produce large cell colonies were observed. Proapoptotic activity was evident from the DNA fragmentation and staining methods. ZR3 blocked the cells in the G2/M phase of the cell cycle. In vivo studies suggested the antiproliferative and proapoptotic activities of ZR3. ZR3 exhibited antiangiogenic properties in vivo. Conclusion: This study confirmed the role of Z. rhetsa partially purified proteins as a potential proapoptotic and antiangiogenic agent against the MCF-7 cell line.

Keywords: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-diphenyl tetrazolium bromide, Anticancer, chorioallantoic membrane, in vivo, MCF-7, vascular endothelial growth factor, Zanthoxylum


How to cite this article:
Naik Parrikar PD, Srinivas BK, Krishnappa DK, Jayarama S. Apoptosis-inducing and antiangiogenic activity of partially purified protein from the pericarp of Zanthoxylum rhetsa in vitro and in vivo. Phcog Mag 2021;17, Suppl S1:96-104

How to cite this URL:
Naik Parrikar PD, Srinivas BK, Krishnappa DK, Jayarama S. Apoptosis-inducing and antiangiogenic activity of partially purified protein from the pericarp of Zanthoxylum rhetsa in vitro and in vivo. Phcog Mag [serial online] 2021 [cited 2021 Jun 19];17, Suppl S1:96-104. Available from: http://www.phcog.com/text.asp?2021/17/5/96/318031



SUMMARY

  • Zanthoxylum rhesta partially purified protein could be a potential proapoptotic and antiangiogenic agent and could be easily included in the cancer treatment strategies due to its edible and nontoxic nature




Abbreviations used: ZRPPP: Zanthoxylum rhetsa partially purified proteins; EAC: Ehrlich ascitic carcinoma; CAM: Chorioallantoic membrane; MVD: Microvessel density; H and E staining: Hematoxylin and eosin staining; NCCS: National Center for Cell Sciences; FACS: Fluorescence-activated cell sorter; VEGF: Vascular endothelial growth factor; NCI: National Cancer Institute.


   Background Top


Cancer cases have been predicted to increase exponentially with an almost 60% hike by 2040.[1],[2] The current treatment strategies mostly rely on chemotherapy employing cytotoxic drugs which target cancer cells. Unfortunately, this mode of treatment is also associated with side effects and other secondary complications, including adverse effects on the nervous system, immune suppression, etc.[3],[4]

Vinca alkaloids such as vincristine and vinblastine paved the road for the use of phytochemicals for cancer treatment.[5] Ever since plants and their bioactive compounds have been the preferred choice for the development of anticancer drugs, various plant extracts and bioactive compounds are rich in proapoptotic and antiangiogenic compounds. Phytochemicals and secondary metabolites such as flavonoids and phenolic compounds have been communicated to be effective against cancer.[6]

Zanthoxylum rhetsa, an aromatic spice normally used as a flavoring agent in Asian cooking, is widely dispersed in the tropical and subtropical regions including India, China, and Malaysia.[7] Various parts of this tree have been studied for myriad of medicinal applications; conventionally, Z. rhetsa is used as an aromatic, astringent, antimicrobial, antiseptic, and antidiabetic agent. It has been used in the treatment of cholera, dermatosis, toothache, and snake bites. The Kannikar tribe in Tamil Nadu, India, uses the prickly thorns to alleviate breast pain in breastfeeding mothers, the Adi tribe of Arunachal Pradesh, India, uses the plant shoot as a vegetable, while people from the coastal region of Maharashtra, India, use the leaf extract as wormicide.[8]

Compounds obtained from Z. rhetsa bark include evodiamine, dihdrovicine, rhestine, and chelerythine, while from fruits include dictamine and arboline.[9] Work involving the study of the cytotoxic effect of quinolone, terpene alkaloids isolated from the root bark of Z. rhetsa against stomach cancer cell lines SCL, SCL-6, SCL370 6, SCL-9, Kato-3, and NUGC-4 showed weak cytotoxicity.[10] The cytotoxic effects of the isolated compounds on mouse melanoma cells, B16-F10, were demonstrated in a study involving the extraction of tetrahydrofuran lignans from Z. rhesta bark. Kobusin showed a high percentage of cell death with an IC50 value of 112.2 g/mL.[11] Volatile oils from Z. rhetsa fruits have been prominent in exhibiting strong cytotoxic activity against lung cancer cell line H460.[12] Alkaloids from conical prickles on the stem bark of Z. rhetsa have been investigated for cytotoxicity against colon cancer cell line SW-480, cervical cancer cell line HeLa, and breast cancer cell lines SKBR3 and MDA MB 231.[9] Studies have been undertaken to analyze the anticancer activity of various phytochemicals from the different parts of Z. rhetsa.[13] However, fruit pericarps that are usually used in culinary preparations are still not much focused upon. The edible nature of Z. rhetsa fruits adds advantage as it can be easily incorporated into the treatment regimen without causing secondary complications.

The current study aims at understanding the proapoptotic and antiangiogenic activity of the partially purified proteins from Z. rhetsa fruit pericarp both in vitro and in vivo.


   Methods Top


Chemicals and reagents

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-diphenyl tetrazolium bromide (MTT), acridine orange, ethidium bromide, and trypan blue were purchased from SRL, India.

Cell lines and culture medium

Breast cancer cell lines (MCF-7, MDA MB 231), human cervical cancer cell line (HeLa), and human colon cancer cell line (HCT-116) were procured from National Centre for Cell Sciences (NCCS), Pune, India, and cultured in DMEM and McCoy's 5A medium respectively, supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 μg/mL) in humidified condition with 5% CO2 at 37°C till confluency. Then trypsinized with 0.2% trypsin, 0.02% EDTA, 0.05% glucose in phosphate-buffered saline (PBS). Trypsin and antibiotics were acquired from Gibco and Invitrogen Life Technologies (Paisley, UK), respectively. Swiss albino mice were procured from a private firm, Sri Venkateshwara Enterprises, Bangalore, Karnataka.

Sample collection and protein extraction

Dried fruits of Z. rhetsa were collected from the Western Ghats region of India, deseeded, and ground to a fine powder. The plant was authenticated by the Department of Botany, University of Mysore, Mysore, India (voucher specimen number: UOMBOT19ZR9). Pulverized powder of Z. rhetsa pericarp (25 g) was subjected to continuous agitation in 200 mL of PBS, pH 7.0 at 4°C for 4 h. This extract was filtered and centrifuged at 10,000 rpm for 10 min at 4°C.[14] The supernatant was used for ammonium sulfate precipitation at different concentrations, 0%–40%, 40%–60%, and 60%–80% and was labeled as ZR1, ZR2, and ZR3, respectively, dialyzed against PBS with dialysis membrane of molecular cutoff 2.5 kDa (Sigma Aldrich), pH 7.0, 4°C. These dialyzed fractions were concentrated by lyophilizing with BioTron vacuum freeze dryer and used to evaluate the cytotoxicity.

Analysis of protein

Lowry's method was used for determining the concentration of proteins with bovine serum albumin (BSA) as the standard reference with trivial modifications. Proteins were separated following the Laemmli method on 12.5% SDS-PAGE. The resultant gel was stained with Coomassie Brilliant Blue R-250. A comparison was made with a standard protein molecular marker.[15],[16]

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H- diphenyltetrazolium bromide assay

Cytotoxicity of the protein samples (ZR1, ZR2, and ZR3) was analyzed by MTT assay. Cells (1 × 106) were inoculated per well in a 96-well microtiter plate and subjected to different concentrations of the sample (0, 10, 20, 40, 80, 160, and 320 μg) followed by 24-h incubation at 37°C, 5% CO2 atmosphere. Absorbance was read at 590 nm in a multimode reader, Infinite M200 PRO, TECAN.[17] ZR3 was renamed as Z. rhetsa partially purified protein (ZRPPP) and this name was used for further assays.

Colony formation assay

MCF-7 cells were cultured 400 cells/well in a 6-well plate and treated with ZRPPP (20 μg/mL and 40 μg/mL) for 24 h. Colonies surviving the treatment were fixed with methanol–acetic acid solution and fixed with 0.4% crystal violet. Colonies were observed and photographed at × 10.[18]

Wound healing assay

MCF-7 cells (1 × 106) were allowed to confluent; a scratch of 1 mm size was made using a microtip followed by the removal of media containing cellular debris. Fresh medium along with sample ZRPPP (20 μg/mL and 40 μg/mL) was added and incubated for 48 h. Changes in the gap were noted and photographed at ×10 using the CatCam 130 microscope camera.[19]

Animal experiments

Swiss albino female mice, 6–8 weeks old weighing around 25 ± 1.5 g, were harbored as per the standard laboratory conditions and fed an animal chow diet with ad libitum water throughout the experiment at room temperature with good ventilation and light/dark cycle of 12 h. Animals were acclimatized for 1 week before the initiation of the animal experiments.[20] These experiments were by the regulations set by the Institutional Animal Ethics Committee (IAEC, Approval No: BCP/IAEC/EXTP/04/2018), Bharathi College of Pharmacy, Bharathi Nagara, Mandya district, Karnataka, India.

In vivo studies on tumor development and treatment

Ehrlich ascites carcinoma (EAC) cells are used for the induction of tumor development due to their native mouse origin, highly transplantable nature, short life span, and 100% malignancy. A predetermined number (1 × 106) of viable cells collected from the donor mice were injected into the peritoneal cavity of the recipient mouse and were left to multiply.[21],[22] After 10 days of inoculation, cells were withdrawn from the donor mouse and diluted with saline, and approximately 5 × 106 cells per animal were administered intraperitoneally.[23],[24] These animals were utilized for further experiments. Growth was monitored daily by recording the body weights of the mice until the end of the experiment. Cell growth is marked by the increase in weight and the abdominal swelling in mice. Mice were divided into two groups, Group I served as a control group (EAC-bearing, n = 6), and Group II received the treatment with ZRPPP 25 mg/kg bodyweight of the mouse (n = 6). On transplantation on the 7th day, three alternate day doses of the sample were administered intraperitoneally. The survivability of the animals was documented separately.[18],[25]

Apoptotic studies

Giemsa staining

MCF-7 cells (in vitro) and EAC cells (in vivo) were trypsinized from treated and control culture plates and fixed with 3:1 methanol acetic acid solution onto glass slides and stained with 0.1% Giemsa stain. Cells were observed under a light microscope and photographed at ×20 using the CatCam 130 microscope camera.[17]

Fluorescence study

Acridine orange–ethidium bromide staining

Acridine orange–ethidium bromide staining was performed as per previously reported protocols, 1 × 105 MCF-7 cells and EAC cells were treated with ZRPPP (20 μg and 40 μg) for 24 h, and these cells were stained with acridine orange–ethidium bromide (10 μg/mL) for 2 min with gentle mixing. This suspension was smeared onto a glass slide and examined under a fluorescence microscope and photographed at ×40 (Carl Zeiss Axio Imager A2).[26]

DNA fragmentation assay

Genomic DNA was isolated using the phenol–chloroform–isoamyl alcohol method described earlier, MCF-7 cells and EAC cells were centrifuged, and the pellets were processed for DNA isolation. The obtained DNA was electrophoresed on 2% agarose gel and visualized under Gel Doc System (Bio-Rad Universal Hood II) and documented.[19]

Annexin V staining

MCF-7 cells (1 × 105 cells/mL cells) were cultured in 24-well plates and treated with ZRPPP (20 μg and 40 μg) and control for 24 h, and the cells were harvested and pelleted. The obtained pellet was resuspended in 50 μL binding buffer containing 0.5 μL Annexin V-FITC followed by dark incubation at 4°C for 30 min. Propidium iodide (PI), 50 μL/mL was added to the binding buffer followed by 5 min incubation, and cells were analyzed by flow cytometry.[27]

Fluorescence-activated cell sorting

The monolayer of the cells was trypsinized and centrifuged to obtain a cellular pellet and washed with PBS; cell pellet was fixed with sheath fluid followed by drop by drop addition of 70% chilled ethanol and further maintaining cells at 4°C for 30 min or overnight. On fixation, cells were centrifuged at 2000 rpm at 4°C for 5 min. The obtained cell pellet was washed and resuspended in sheath fluid containing 0.05 mg/mL PI and 0.05 mg/mL RNase followed by 30 min incubation in dark at room temperature. The cell percentage arrested at various phases of the cell cycle was determined using fluorescence-activated cell sorter caliber (BD Biosciences, San Jose, CA).[17],[28]

Angiogenic studies

Vascular endothelial growth factor enzyme-linked immunosorbent assay

Ascitic cells from treated and control mice were diluted to 1:10, and 100 μL of this was used for coating 96-well microtiter plates with a coating buffer overnight at 4°C. These wells were washed and reincubated with anti-vascular endothelial growth factor (VEGF)165 antibodies. Further this reaction mixture was incubated with secondary antibody (goat anti-rabbit IgG) tagged with alkaline phosphatase. This reaction mixture was incubated for 2 h at room temperature; to this, 100 μl of p-nitrophenyl phosphate was added, and the absorbance was measured at 405 nm (Thermo Scientific Varioskan Flash Multimode Reader).[16],[23]

Chorioallantoic membrane assay

In ovo chorioallantoic membrane (CAM) assay was executed to determine the antiangiogenic properties of ZRPPP on the CAM of fertilized eggs as per the methods described earlier. CAM was photographed.[29] Microvessel density (MVD) was counted in ten fields of vascularized areas under high power, and the mean MVD counted was recorded from the peritoneal linings of control and treated mice. Peritoneal linings obtained were fixed in formaldehyde and blocked into paraffin for sectioning and hematoxylin and eosin (H and E) staining.

Histopathological analysis by hematoxylin and eosin staining

The liver and spleen were aseptically obtained from mice sacrificed by the cervical dislocation.[30],[31] Histopathological analysis was done on the liver, spleen, and intraperitoneal tissues. In brief, these tissues were fixed in 4% paraformaldehyde and fixed with paraffin wax; 10 μm sections were made using a microtome. These sections were stained with H and E stain and were observed under a low-power light microscope.[17],[19]

Statistical analysis

All the values were indicated as the mean ± standard error of the mean for both the control and experimented studies. Statistical analyses were performed using ANOVA, Student's t-test, and Kaplan–Meier analysis using GraphPad Prism 8.0.2 (263) (GraphPad Software Inc, California).


   Results Top


Zanthoxylum rhetsa partially purified proteins show cytotoxic activity

Proteins obtained from Z. rhetsa dialyzed fractions – 0%–40%, 40%–60%, and 60%–80% – showed promising cytotoxic activity against the different cell lines of various origins such as human and murine mammary cancer, human colon cancer, and human cervical cancer. 0%–40% (ZR1) fraction showed IC50 values of 76.95 μg/mL, 76.99 μg/mL, 97.45 μg/mL, and 88.96 μg/mL for HeLa, HCT-116, MDA MB 231, and MCF-7, respectively; the next fraction 40%–60% (ZR2) showed IC50 values of 68.95 μg/mL, 70.31 μg/mL, 79.98 μg/mL, and 68.94 μg/mL for HeLa, HCT-116, MDA MB 231, and MCF-7, respectively. The last fraction 60%–80% (ZR3) IC50 values were found to be 23.3 μg/mL, 50.6 μg/mL, 71.16 μg/mL, and 21.5 μg/mL HeLa, HCT-116, MDA MB 231, and MCF-7, respectively [Figure 1]a,[Figure 1]b,[Figure 1]c,[Figure 1]d. Among the three dialyzed fractions, 60%–80% fraction showed higher inhibitory percentage against MCF-7 cell line. Thus, ZR3 fraction was chosen for further assays. This fraction showed the presence of four proteins in 12.5% SDS-PAGE. These proteins had molecular weights ~ 45 kDa, ~31 kDa, ~24 kDa, and ~8 kDa [Supplementary Figure 1]. ZRPPP will be used to represent the protein fraction ZR3 for results henceforth.
Figure 1: In vitro cytotoxic effect of ZR1, ZR2, and ZR3 on (a) HeLa cells, (b) HCT116, (c) MDA MB 231, (d) MCF-7

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Zanthoxylum rhetsa partially purified proteins inhibits cancer cell proliferation in vitro and in vivo

Based on the IC50 values (IC50 >50 μg/mL), MCF-7 cell line was stipulated for further studies. Long-term cytotoxicity was evaluated based on the changes in the reproductive integrity of cells by colony formation assay. At 20 μg concentration, ZRPPP showed ~ 66% survival and ~53% at 40 μg concentration as compared to the control [Figure 2]a and [Figure 2]b. The interactive property of the cells needed to fabricate contact with surrounding cells and extracellular matrices is displayed by proliferating cells, and this is exploited in wound healing assay. Reduction in the cellular migration to heal the wound created was observed in treated cells as compared to control cells which filled the gap [Figure 2]c and [Figure 2]d.
Figure 2: ZRPPP inhibits cancer cell proliferation in vitro and in vivo. (a) Colony formation assay (MCF-7). (b) Graph representing percentage of colony formation. Statistical significance: **P < 0.001, ****P < 0.0001. (c) Wound healing assay (MCF-7 cells), (d) Graph representing migration of cells. Statistical significance: **P < 0.001, ****P < 0.0001. ZRPPP: Zanthoxylum rhetsa partially purified proteins

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A dose of 25 mg/kg bodyweight of the animal was administered intraperitoneally to the animals. On completion of the tenure of dose, hematological and serum parameters were analysed. [Supplementary Table 1]. Morphological comparisons between the treated and control mice showed a lack of secondary complications and rendered the compound under study nontoxic for mice treatment. Dynamics in the growth of intraperitoneally inoculated EAC cells were monitored pre- and post-treatment till the animals were sacrificed. It showed a decrease in the tumor weight evident from the decrease in the bodyweight of the ZRPPP-treated animals as compared to the control animal [Figure 3]a and [Figure 3]b.
Figure 3: ZRPPP reduces EAC cell proliferation in vivo. (a) Morphological changes in mice. (b) Graph of bodyweight of treated (25 mg/kg body weight) and control mice. (c) Cell density of viable cells in treated and control mice. (d) Changes in the ascetic volumes of control and treated mice. (e) Kaplan–Meier graph of survivability (n = 6) Statistical significance: * P < 0.05, **P < 0.001, ****P < 0.0001. ZRPPP: Zanthoxylum rhetsa partially purified proteins; EAC: Ehrlich ascitic carcinoma

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A comparative decrease in the cell number was observed from the cell count analysis undertaken with a trypan blue dye exclusion study [Figure 3]c. Reduction in the tumor load was also evident from the comparative decrease in the ascitic fluid, 12.7 mL in control, whereas ascetic load decreased to 3.4 mL for the treated mice, thus indicating the cytotoxic effect of ZRPPP [Figure 3]d. Treatment also reflected enhancement in the average survival from 15 days to 30 days [Figure 3]e. Histopathological analysis of liver, spleen, and kidney of treated and EAC-bearing animals showed slight restoration of the normal histological features in the treated organ sections [Figure 4]a,[Figure 4]b,[Figure 4]c,[Figure 4]d,[Figure 4]e,[Figure 4]f.
Figure 4: Histomorphological analysis of control and ZRPPP (25 mg/kg bw) treatment. (a) Morphological changes in liver. (b) Histopathological analysis of liver. (c) Morphological changes in spleen. (d) Histopathological analysis of spleen. (e) Comparison of changes in weight of control and treated mice liver. (f) Comparison of weights of control and treated mice spleen. Statistical significance: * P < 0.05, **P < 0.001, ****P < 0.0001. ZRPPP: Zanthoxylum rhetsa partially purified proteins

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Zanthoxylum rhetsa partially purified proteins induces apoptosis and DNA fragmentation in vitro and in vivo

Staining of the cells with Giemsa showed the typical formation of apoptotic bodies in the cells treated with ZRPPP indicating the proapoptotic action of ZRPPP also this fact was further validated by acridine orange–ethidium bromide staining which clearly showed yellow-green fluorescence in treated cells indicating apoptosis [Figure 5]a and [Figure 5]b. The disintegration of DNA is one of the hallmarks of apoptosis; DNA of MCF-7 cells pretreated with ZRPPP showed the fragmentation pattern of DNA as compared to the intact linear DNA in untreated cells, indicating the apoptotic ability of ZRPPP [Figure 5]c. Similar outcomes were evident in vivo, EAC cells treated with 25 mg/kg ZRPPP per animal; staining of the treated and the control EAC cells showed the typical apoptotic behavior in Giemsa and acridine orange–ethidium bromide staining. Cellular DNA fragmentation was seen in treated mice as compared to the linear DNA in control EAC-bearing mice [Figure 6]a and [Figure 6]b. Treated in vivo EAC cells showed apoptotic bodies which are prominent feature of apoptosis. DNA fragmentation was also observed the treated cells [Figure 6]c.
Figure 5: Apoptotic activity of ZRPPP in vitro (MCF7 cells). (a) Giemsa staining, (b) Acridine orange–ethidium bromide, (c) DNA fragmentation in treated cells (Lane 1: control, Lane 2: treated). Statistical significance: * P < 0.05, **P < 0.001, ****P < 0.0001. ZRPPP: Zanthoxylum rhetsa partially purified proteins

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Figure 6: Apoptotic activity of ZRPPP in vivo. (a) Giemsa staining, (b) acridine orange-ethidium bromide, (c) DNA (MCF-7 cells) fragmentation assay. ZRPPP: Zanthoxylum rhetsa partially purified proteins

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Zanthoxylum rhetsa partially purified proteins induced apoptosis and arrested the cells

ZRPPP induced apoptosis, indicated by the percentage of apoptosis in flow cytometry. This was exhibited by the increase in the apoptotic percentage of the treated cells as compared to the control cells. Treated cells showed 0.35% of early apoptotic and 8.05% of late apoptotic cells for 20 μg/mL concentration. The percentage of early apoptotic cells was 0.04 percent and 17.28 percent of late apoptotic cells were found in the treated cells at a concentration of 40 g/mL-1, compared to 0.7 percent of early apoptotic cells and 4.36 percent of late apoptotic cells in the untreated cells [Figure 7]c and [Figure 7]d. In conjunction with Annexin-V apoptosis assay, cell cycle distribution analysis showed that ZR impels cell cycle arrest in the G2/M phase at 20 μg/mL, whereas at 40 μg/mL exposure for 24 h, ZR showed an increase in arrested cells in S phase and G2/M phase of MCF-7 cell line [Figure 7]a and [Figure 7]b.
Figure 7: Cell cycle arrest and apoptosis in ZRPPP-treated MCF-7 cells. (a) Cell cycle arrest. (b) Graph for distribution of cell cycle phases. (c) Apoptosis in control and ZRPPP-treated cells. (d) Graph for percentage of apoptotic cells. Statistical significance: * P < 0.05, **P < 0.001, ****P < 0.0001. ZRPPP: Zanthoxylum rhetsa partially purified proteins

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Zanthoxylum rhetsa partially purified proteins restricts angiogenesis in vivo and in ovo

Peritoneal angiogenesis in ZRPPP-treated and control EAC-bearing mice showed a great decline in the treated animal; further, there was a reduction in the MVD in the treated mice as compared to the control mice. These facts were validated by treating the Swiss albino mice-bearing EAC cells with ZRPPP. The peritoneal tissue from the treated mice was subjected toH and E staining showed a reduction in MVD. Observation of the peritoneal angiogenesis showed a comparative reduction in the angiogenesis in the treated animal as compared to the control animal peritoneum. [Figure 8]a,[Figure 8]b,[Figure 8]c,[Figure 8]d. The antiangiogenic property of ZRPPP was supported by CAM assay which indicated a reduction in blood vessel formation [Figure 8]e and [Figure 8]f. Reduction in the VEGF level as compared to the control indicated the antiangiogenic role of ZRPPP [Figure 8]g.
Figure 8: Antiangiogenic activity of ZRPPP in vivo. (a) Peritoneal angiogenesis in normal, control, and treated mice. (b) Representative graph for MVD in normal, control, and treated animal peritoneum. (c) Histopathological analysis of MVD of peritoneal tissue. (d) Graphical representation of changes in MVD in the MVD H and E staining. (e) Reduction in angiogenesis in the CAM in ovo. (f) Representative graph of MVD in normal, control, and ZRPPP 25 μg in CAM. (g) Changes in VEGF levels in control and treated ascites. Statistical significance: * P < 0.05, **P < 0.001, ****P < 0.0001. ZRPPP: Zanthoxylum rhetsa partially purified proteins; CAM: Chorioallantoic membrane; MVD: Microvessel density; H and E staining: Hematoxylin and eosin staining; VEGF: Vascular endothelial growth factor

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   Discussion Top


Spices have been savored since time immemorial as part of cuisines for their flavors and aroma. Moreover, ancient literature reflects their applications as medicines and medicinal preparations. The last few years has seen an immense increase in the prevalence of chronic diseases and also the associated surge in the cost of healthcare commodities. Statistics of 2018 by the World Health Organization reveals cancer as the second leading cause of death globally contributing to 1 in 6 deaths. Moreover, one-third of deaths from cancer are due to dietary risks and lifestyle changes.[32] Complementary and alternative medicine studies include the group of modalities in the treatment regimen which are not part of the standard treatment; thus, the use of the anticancer potential of routine dietary constituents such as fruits, vegetables, herbs, and spices should be considered. Various epidemiological and preclinical studies have inferred the importance of anticancer characteristics of culinary herbs and spices.

Many of the currently available drugs are plant-based drugs, plant peptides, and proteins have turned out to be a critical source of biological compounds that have exhibited bioactivities and can be exploited as a drug.[33] The current study aims at studying the proapoptotic and antiangiogenic potential of partially purified proteins obtained from Z. rhetsa pericarp. Z. rhetsa is a member of Rutaceae family widely dispersed throughout the tropical regions of the world. This family comprises more than 1500 species.[34] This fragrant plant is known to have biological properties such as antioxidant, antimicrobial, and antifungal; further, it has been analyzed for anticancer properties by some workers. Various parts and components from Z. rhetsa have already been evaluated for their cytotoxic and anticancer activities. As Z. rhetsa seed pericarp is commonly used as a spice in the coastal regions of Western India; a comprised study of its cytotoxicity and related anticancer activity can help in classifying it as complementary and alternative medicine. This study exploited the very fact that pericarp of Z. rhetsa is used in culinary preparations and its associated ethnomedicinal properties which could be easily brought into regular usage.

Prevalent anticancer treatments are associated with side effects that ill-treat the normal cells along with the target cells, thus leading to secondary complications associated with synthetic drugs. Thus, the scientific community has been in constant search of anticancer drugs of natural and dietary origin in such a way that could help cancer patients to survive and improve the quality of life. Literature has reflected several studies that have focused on the usage of plant-derived proteins as potential anticancer products that have no or mild secondary complications. This study is aimed at the evaluation of the partially purified ZRPPP, which comprises four different proteins ranging from ~ 45 kDa to 8 kDa. ZRPPP has shown promising cytotoxic activity against MCF-7 breast cancer cell lines as compared to other cell lines at a lower concentration.

The study also reflected the antiproliferative nature of ZRPPP by resulting in a comparative reduction in the formation of large cellular colonies to be formed upon treatment with ZRPPP. Treatment also resulted in the failure of migration of cells, thus failing to fill the wound or the gap created. DNA fragmentation resulted in the death of the cells that concluded the proapoptotic activity of ZRPPP.

Colony formation by cells is an indication of the reproductive ability of the cell; it determines the ability of each cell to sustain unlimited division. On exposure to the treatment, cells that can express the proteins and DNA undergo one or two cycles of mitosis but cannot produce a large number of progenies and hence considered as reproductive death.[35],[36] Yet another and most evident property of cancer cells is invasive proliferation and migration.In wound healing assay, migration of cells toward the "wound" is observed thus filling the gap formed as a result of the wound.

The study was further validated in vivo using a Swiss albino mice model inoculated with EAC cells. These cells are one of the most suitable models, because of their mouse origin and easy transplantabilty. The study clearly showed a reduction in the tumor load evident from the changes in the body morphology of the tumor-bearing mice as compared to treated mice bearing the EAC cells. ZRPPP was found to be nontoxic as supported by the biochemical parameter studies. In vivo study was supported by morphological and histological changes in the organs such as liver and spleen of control and treated animals. This showed negligible changes upon treatment. Further, there was a moderate improvement in the survival of the treated EAC-bearing mice as compared to the EAC-bearing mice.

The study was supported with the proapoptotic activity of ZRPPP by staining techniques such as Giemsa and acridine orange–ethidium bromide which exhibited the comparison between the control and the treated cells. A similar study was carried out in vitro against the MCF-7 cell line at 20 μg and 40 μg concentrations which indicated the formation of apoptotic bodies and ethidium bromide intercalation with the nuclear material of the apoptotic cells. Acridine orange being membrane-permeable, the apoptotic cells are seen to have a dual-stained appearance.

Moreover, the DNA fragmentation pattern presented by treated EAC and MCF-7 cell lines in vivo and in vitro, respectively, indicated induction of apoptosis. The proapoptotic activity of ZRPPP was also supported by flow cytometric study and cell cycle analysis which established the proapoptotic nature of ZRPPP. ZRPPP arrested the treated MCF-7 cells in the G2/M phase of the cell cycle, thus showing its efficiency as a potent cell cycle arresting product.

The in vitro and in vivo pieces of evidence suggested the antiproliferative and proapoptotic activity of ZRPPP, thus giving a new outlook to the edible nature of Z. rhetsa.

Angiogenesis is another factor that is prominent in cancer; sprouting of new blood vessels is very much essential for tumor growth; hence, it becomes a riveting target for analysis of compounds under study. The hindrance of angiogenesis becomes one of the probable mechanisms which could be employed as an anticancer strategy. Changes in the neovasculature in the peritoneal tissues of EAC-bearing mice indicate the efficacy of the compound. Treatment of EAC-bearing mice with 25 mg/kg body weight with ZRPPP resulted in a reduction in the sprouting tendency of blood vessels as compared to the control mice which showed extensive angiogenesis.

VEGF secreted by the EAC cells is the growth factor that promotes angiogenesis; thus, quantification of secreted VEGF becomes one of the important factors that determine the effect of the compound under study on angiogenesis. This study showed a decrease in the secreted VEGF, thus indicating the role of ZRPPP. Further, in ovo study involving CAM assay indicated a decrease in blood vessel formation. The comparatively reduced MVD also indicated the antiangiogenic nature of ZRPPP.

Moreover, there was a restoration of organ morphology in the treated animal and the H and E staining showed negligible usual treatment associated with secondary complications. Although many studies have been attempted to study the role of Z. rhetsa as an anticancer agent, limited focus has been given to the edible fruit pericarp of the plant. As the fruit pericarp is routinely used culinary preparation, the major findings of this study further strengthened existing literature supporting anticancer properties of Z. rhetsa. In brief, ZRPPP can serve as a potent proapoptotic and antiangiogenic product that can be used as an anticancer agent.


   Conclusion Top


Z. rhetsa pericarp partially purified proteins showed cytotoxic activity against the human cancer cell lines in vitro. The fraction ZRPPP showed cytotoxic and antiproliferative activity against the MCF-7 cells in vitro and EAC cells in vivo in Swiss albino mice. ZRPPP was also found to have proapoptotic role and arrested the MCF-7 cells in G2/M. Moreover, ZRPPP exhibited antiangiogenic effect in vivo and in ovo. Thus, this study suggests the potential of partially purified proteins from Z. rhetsa fruit pericarp (ZRPPP) as a proapoptotic and antiangiogenic agent in cancer treatment.

Acknowledgements

The authors acknowledge the Teresian Research Foundation and the P. G. Department of Biotechnology, Teresian College (Affiliated to the University of Mysore), for providing animal cell culture facility. The authors would like to thank the Department of Pharmacy, Bharathi College of Pharmacy, KM Doddi, Mandya, Karnataka, for providing the animal house facility.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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