Lupine Publishers | A Brief Review on Phytochemical and Pharmacological Aspects of Andrographis Paniculata

 Lupine Publishers | LOJ Pharmacology & Clinical Research

 

Introduction

Since the beginning of civilization, medicinal plants have been an intrinsic component of human life [1]. The conservation of ethnobotanical knowledge as part of living culture and practice between communities and the environment is essential for biodiversity conservation. The information about medicinal plants gains from various medicinal systems such as Unani, Siddha, and Ayurveda [2]. The traditional system of medicine belongs to the traditions of each country and has been passed over from generation to generation. Understanding the dynamics of traditional local knowledge of medicinal plants is important for their medicinal properties is now being developed as a source of scientific research to prove the effect of plants and generate new therapeutic resources. Medicinal plants are considered as a backbone of traditional medicine (WHO) as well as most modern medicine is also derived from medicinal plants i.e. aspirin. The medicinal plant having a rich source of components that can be used to develop and synthesize drugs. About 3.3 billion people in developing countries depend on medicinal plants on a regular basis, WHO estimated that about 80% world population rely on the medicinal plant for their primary health care. Further more, worldwide 42% of 25 top-selling drugs marketed are either directly obtained from natural sources or entities derived from plant products [3]. The quality of traditional medicine is determining its active substances produced by the plant. Andrographis paniculata is one of the important medicinal plants that is utilized throughout the world [4]. A.paniculata is an herbaceous plant of the Acanthaceae family. It is widely distributed in Southeast Asia, India, and tropical as well as in subtropical Asia. A.paniculata is also known as the “King of Bitters” since it has a highly bitter taste in all parts of the plant body [5]. Furthermore, A. paniculata is known as “Kalmegh” in India, “Chuan-Xin-Lian” in China, “Fah Tha Lai” in Thailand, “Hempedu Bumi” in Malaysia, “Senshinren” in Japan, and “green chiretta” in Scandinavian nations [6]. A.paniculata is one of the most widely used plants in Ayurvedic and Unani medicine [4]. Traditionally, A.paniculata was used in the treatment for snakebite, fever, bug bite, diabetes, malaria, and dysentery [7]. Moreover, A.paniculata is also used in the combination with other herbs and health care treatment. It is found that A.paniculata is used in more than half of the herbal formulations commercialized in India for he patic diseases [8]. Many scientific studies also have been reported regarding the medicinal properties possessed by the A.paniculata, most of which are based on traditional knowledge (Table 1). Phytochemical investigations have revealed that A. paniculata contains a wide range of chemicals. In addition, experimental evidence also reported that A.paniculata has a broad spectrum of pharmacological activity including anti-bacterial, antidiarrheal, anti-inflammatory, antiviral, antimalarial, anticancer, antimalarial, hepatoprotective, etc. In this review, we briefly discuss ethnobotanical uses, phytochemistry, and recent scientific finding pharmacological activity of the A.paniculata [6].

Table 1: Taxonomical classification of Androgrphis paniculata.

Botanical Description and Habitat

A.paniculata is native species of India, China, and Taiwan. But it is also found in Southeast Asia, tropical and subtropical Asia as well as few other nations such as Malaysia, Indonesia, Vietnam, Sri Lanka, Laos, Cambodia, Pakistan, Myanmar, and the Caribbean islands [9]. Especially, in India A.paniculata are found in Karnataka, Andhra Pradesh, Tamil nadu, Uttar Pradesh, and Madhya Pradesh. Also cultivated in Assam and West Bengal to some extent. In addition, A.paniculata are found in different habitats including forests, farms, plains, hill slopes, dry and wetlands, and wastelands [10]. A.paniculata is bitter in test, an annual herb that is abundantly branched which grows up to a height of 3.-110 cm in a humid, shady area. It has glabrous leaves that are 8.0 cm long and 2.6 cm wide, little white flowers that are rose-purple or light pink, spots on the petals, and corolla with hairs. The stem was found to be dark green in color, 0.4-1.0 m tall, 2-6 mm in diameter, quadrangular with longitudinal furrows and wings on the angles of the younger part [11] as shown in Figure 1.

Figure 1: Andrographis paniculata morphology.

Traditional Uses of Andrographis Paniculata

A.paniculata play vital importance in the Ayurvedic, Siddha, and traditional medicine systems in India [12]. For centuries, the leaves and roots of A.paniculata have been used to treat a wide range of health problems in Asia and Europe. However, the entire plant is also utilized for specific uses [13]. The plant known as “Kalmegh” in Ayurvedic literature is an essential element in the majority of Ayurvedic remedies and is officially recognized by Ayurvedic pharmacopeia. Moreover, it is used as an aperient, emollient, astringent, anti-inflammatory, diuretic, anthelmintic, carminative, and antipyretic in the Unani system of medicine [14].In India, tribal groups used this herb to cure a number of diseases such as antidote against snake bites, Banded Krait and Russell’s viper, etc. [14]. The tribal of Kheria, Khatra, Moora, and the Santal region of Bankura district, West Bengal, India utilizes an infusion of the entire plant to treat fever [15]. The extracted juice from A.paniculata leaves, alone or combined with cloves, cinnamon, and cardamom is used as a cure for flatulence, loss of appetite, griping, diarrhea in children, and irregular stool. In India during the influenza epidemic in 1919, A.paniculata was shown to be highly effective in reducing the disease progression [16]. It was also utilized by ancient Chine’s physicians to treat inflammatory diseases, colds, laryngitis, and fever, hepatitis, pneumonia, respiratory infections, tonsillitis, sores, pelvic snake bites, herpes zoster and it has been characterized as a cold property herb [13] to remove toxins and body heat. The decoction of fresh leaves of A.paniculata is used as an antihypertensive and antidiabetic in Malaysian folk medicine. Furthermore, it is advised to use it in cases of leprosy, scabies, gonorrhea, boils, chronic and seasonal fevers, and skin eruptions, due to its “blood purifying” purifying properties [4].

Phytochemistry

The aerial part (leaves and stems) of A. paniculata contains major active phytochemicals [17]. According to the survey of the literature, andrographolide is the major bioactive compound found in the A.paniculata which is a diterpene lactone that is crystalline, colorless, and has a bitter taste [9]. The leaves have the highest concentration of andrographolide about 2.39% whereas the seed has the lowest concentration about 0.58%. The quantity of the phytochemicals varies widely depending on the portion used, locality, time of harvesting, and season (Figure 2). Andrographolides are highest found immediately before the flowering season, then decline progressively [14]. Other lactones compound observed in A.paniculata is 14-deoxy-11-andrographolide, 14-deoxy-11, 12 didehydroandrographolide, andragraphan, andrographon, 14-deoxyandrographolide, neoandrographolide, deoxyandrographiside, andrographosterol, andrographiside etc. A.paniculata also contains Xanthones and quinic acid derivatives in minor concentrations. Moreover, Reddy et a. [18] reported that A.paniculata contains flavone such as 5-hydroxy-7’2’6’-trimethoxyflavone and 23-C terpenoid 14-deoxy-15-isopropylidene-11, 12- didehydroandrograholide and other flavonoid Skullcapflavone I 2’-O-glucoside, 7-Omethylwogonin, 7-Omethyldihydrowogonin and 7-O-methylwogonin 5-O-glucoside as well as diterpenoids such as isoandrographolide 14-deoxy-11, 12 didehydroandrographolide. Rao et al. [19] identified and isolated 5, 7, 20, 30-tetramethoxyflavanone and 5-hydroxy-7, 20, 30-trimethoxy flavone from the A.paniculata. A new labdane type diterpenoid which is andropanolide along with seven known diterpenoids isolated from the methanolic leaves extract of A.paniculata [20].

Figure 2: Chemical structure of major component found in A.paniculata.

Pharmacological Activity of A.Paniculata

Hepatoprotective activity

A.paniculata is widely used as a hepatoprotective and hepatostimulative agent in the Indian traditional medicine system. Traditionally the leaves aqueous extract of A.paniculata is used in the treatment of jaundice and different liver damage. Andrographolide found in the A.paniculata was protective against liver damage in rats and mice induced by carbon tetrachloride. Moreover, Andrographolide also observed significant hepatoprotective against various types of liver damage, induced by galactosamine or paracetamol [21]. The free radical scavenging activity of andrographolide has a significant hepatoprotective effect by lowering lipid peroxidation malondialdehyde product as well as by maintaining glutamic pyruvate transaminase, alkaline phosphatase, and glutathione levels in mice treated with carbon tetrachloride [22]. A.paniculata has been shown antihepatotoxic activity against plasmodium berghei K173-induced hepatic damage in mastomys natalensis [23].

Antibacterial activity

A.paniculata has been shown the antibacterial activity against a wide range of bacterial species. In vitro study found that the aqueous extract of A.paniculata shown antibacterial activity even at the low concentration (25 mg/ml) against E.coli, Shigella, Streptococci, Staphylococcus aureus, and Salmonella [24]. Another similar study leaves aqueous extract of A.paniculata reported against the methicillin- resistant S.aureus and Pseudomonas aeruginosa [25]. Furthermore, A.paniculata is also effective against HAS 1 (herpes simplex virus 1) without any cytotoxicity [26].

Antidiarrheal activity

In developing countries, Diarrhea is one of the most common diseases and it leads to the top ten causes of death among children worldwide [5]. Some drugs such as kaolin-pectin, selenium, loperamide, and bismuth have been used to treat the symptoms. However, it also causes some unfavorable side effects [5]. The study has been found that A.paniculata has significant antidiarrheal properties [27]. According to the study, an ethanolic extract of A.paniculata treated 88.3 % of acute bacillary dysentery cases and 91.3% of acute gastroenteritis cases. Furthermore, andrographolide was found to treat 91% of acute bacillary dysentery cases. The same cure rate of about 91.1% was obtained by providing a compound tablet comprising andrographolide and neoandrographolide in a 7:3 ratio. This was claimed to be more than the cure rate observed with chloramphenicol and furazolidone [28]. A.paniculata was found to be effective in curing patients with acute diarrhea and bacillary dysentery in double-blind investigation [14].

Antimalarial activity

In many tropic and subtopic countries, malaria is still a prevalent disease [14]. A.paniculata was shown to significantly suppress the growth of the Plasmodium berghei [11]. In vitro study of 50% ethanolic extract of the aerial parts (100 mg/g) shown antimalarial activity against plasmodium berghei and in vivo study in rats observed antimalarial activity after intragastric application (1g/kg body weight) [26]. It is suggested that the antimalarial effect of A. paniculata is due to the reactivation of the enzyme superoxide dismutase [5]. Another study has been reported that the crude extract of A.paniculata shown antimalarial activity against the resistant strain of Plasmodium falciparum having an IC50 value of 6mg/ml [29]. In addition, a xanthous compound isolated from the A. paniculata has been shown in vivo antimalarial activity in plasmodium infected Swiss albino mice. The results found that a significant reduction in parasitemia after treatment with a 30 mg/kg dosage [26].

Anticancer activity

Cancer is a set of disorders characterized by abnormal cell proliferation and the ability to penetrate or be spared to other regions of the body. Despite the fact that many diseases have a worse prognosis than most cancers [17]. The extract of A.paniculata having diterpenoid is significantly able to restrict cell proliferation, arrest the cell cycle and induce cell apoptosis of different cancer cells [30- 33]. Treatment of the MDA-MB-231 breast cancer cells with andrographolide extracted from A. Paniculata causes apoptosis of cancer cells and arrests the cell cycle without interfering with the normal growth of cells [34]. The study has been reported that A.paniculata exhibits potent cytotoxic activity against human epidermoid carcinoma of the skin lining of the lymphocytic leukemia cells and nasopharynx [12]. A.paniculata also shown cytotoxic effects against colon cancer cells by suppressing AKT and mTOR phosphorylation levels, resulting in ER stress-induced death [35]. Furthermore, apoptosis in colon cancer cells is induced by the andrographolide via controlling the signaling of pro-apoptotic GRP-78/IRE1/XBP-1/ CHOP [17].

Antidiabetic activity

Diabetes is a metabolic disease characterized by elevated blood sugar levels [36]. According to the WHO reports around 70 million people worldwide suffer from diabetes. Specifically in developing countries, diabetes has become a threat to human health [37]. In vivo study observed that ethanolic extract of A.paniculata exhibit the protective effect in hyperglycemic condition and also protect the tissue damage caused due to oxidative stress in a diabetic rat model produced by streptozotocin [38]. Another study conducted [39] found that oral administration of andrographolide in a dose-dependent manner reduced plasma glucose levels in diabetic rats caused by streptozotocin and wild-type rats.

Conclusion

The entire literature review indicated that Andrographis paniculata exhibits a broad range of phytochemicals and pharmacological activities. The previous study found that A.paniculata contains 50 lactane diterpenoids, 30 flavonoids, and 30 novels phytochemical isolated and identified from A.paniculata. Phytochemical study reveals that Andrographolide is a major compound found in Andrographis paniculata. It has shown a wide spectrum of pharmaceutical activity such as anti-microbial, hepatoprotective activity, anti-inflammatory activity, anti-malarial, anti-diarrheal, anti-diabetic, and cytotoxic activity. The precise information offered as a review here covers the phytochemical and pharmacological information about this plant, providing the muchneeded encouragement to use this plant in creating and sustaining a prospective means of livelihood.

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Lupine Publishers | A Hmsh2 c.2332 T>A Mutation and Membrane-hMSH2/ TCRγδ/NKG2D-Mediated Cytotoxicity of Human Vγ9vδ2 T Cells in Ovarian Serous Cystadenocarcinoma

 Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

Membrane human MutS homologue 2 (mhMSH2) is a well-characterized endogenous ligand for human Vγ9Vδ2 T cells, but its germline gene expression in mhMSH2-overexpressing ovarian serous cystadenocarcinoma (OSC) cells and tissues is unclear. Herein, we discovered a silent hmsh2 c.2332 T>A mutation in the OSC HO8910 cell line (GenBank accession no.MG674653) and identified serval novel soluble forms of hMSH2 protein from its whole cell protein extracts and culture supernatant. The mhMSH2/ TCRγδ/NKG2D-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells were validated in mhMSH2/6-overexpressing SKOV3 cells. Positive expression of cytoplasmic and/or membrane hMSH2/6, cytoplasmic and/or nuclear hMSH3 and complete loss of nuclear expression of hMSH2 was observed in all examined ovarian cancer tissues. The clinical significance of the novel hmsh2 c.2332 T>A mutation, the soluble full-length and truncated forms of hMSH2 proteins, and the complete loss of nuclear hMSH2 protein expression in OSC development and human Vγ9Vδ2 T cell-mediated anti-OSC immunity remain to be further elucidated.

Keywords: Hmsh2; Mutation; Membrane hMSH2; Vγ9Vδ2 T cells; Ovarian serous cystadenocarcinoma.

Abbreviations: hMSH2: Human MutS Homologue 2; MMR: Mismatch Repair; M: membrane; EBV: Epstein-Barr virus; OSC: Ovarian Serous Cystadenocarcinoma; ATCC: American Type Culture Collection; IHC: Immunochemistry; mAb: monoclonal Antibody; FCM: Flow Cytometry; PFA: Paraformaldehyde; FITC: Fluorescein Isothiocyanate; DAPI: 4’,6-diamidino-2-phenylindole; LS: Lynch Syndrome.

Introduction

Human MutS homologue 2 (hMSH2), a critical element of the DNA mismatch repair (MMR) system, is normally localized in the nucleus of host cells and dimerizes with hMSH3 or hMSH6 (hMSH3/6) to form complexes that participate in DNA damage and cell apoptotic signaling. Inherited or acquired defects in the hmsh2 gene or protein lead to dysfunctional correction of errors in DNA synthesis and duplication, the cell cycle and Ig isotype switching of B cells and thus have a close association with the genesis of many types of tumors [1,2]. In our published study, we reported a broad overexpression of membrane hMSH2 (mhMSH2) in a series of epithelial tumor cell lines and Epstein-Barr virus (EBV)- transformed B lymphoid cells [3]. mhMSH2 overexpressed on malignant cells was subsequent characterized as a stress-inducible endogenous protein ligand for human Vγ9Vδ2 T cells, a subset of innate immune cells that play a crucial role in antitumor/antiviral immunity and autoimmunity [3-5]. The induction of ectopic mhMSH2 expression on renal carcinoma cells by oxidative stress via the p38 mitogen-activated protein kinase and c-Jun N-terminal kinase pathways with interleukin-18 promotion was subsequently revealed, but the systemic expression of hmsh2 gene in mhMSH2- overexpressing carcinomas is still unknown [6].
There are 238,700 estimated new cases of ovarian cancer and 151,900 deaths per year worldwide, and thus, this disease is the fifth most common cause of female cancer-related death in the United States in 2018 [7,8]. Among the 4 subtypes of ovarian cancer (high-grade serous, endometrioid, clear cell ovarian carcinomas and non-epithelial subtypes), Ovarian Serous Cystadenocarcinoma (OSC) is the most common and lethal type in women [9]. Previously, we reported the unusual ectopic mhMSH2 expression on the human OSC cell lines HO8910 and SKOV3 and the specific binding of mutated mhMSH2 on SKOV3 cells to synthesized OT3 peptides of human Vδ2 TCR [3,10]. In this work, we further elucidated the hmsh2 gene mutation and ectopic subcellular protein expression in HO8910 cells and the mhMSH2-mediated recognition and cytolytic effects of Vγ9Vδ2 T cells towards SKOV3 cells. The abnormal subcellular distribution of hMSH2/3/6 in different subtypes of ovarian cancer tissues was demonstrated by immunochemistry (IHC).

Materials and Methods

Cell lines, culture medium and tumor tissues

HO8910 cells (OSC) were maintained in RPMI-1640 complete medium (Invitrogen, Shanghai, China). SKOV3 (OSC) and HK-2 (proximal tubular cell line derived from normal kidney) cells were cultured in 10% FBS DMEM/F12 medium (Niuyin, Beijing, China). All cell lines were purchased from the Cell Culture Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences and confirmed to have no mycoplasma contamination before use. Expansion and subset separation of effector human Vγ9Vδ2 T cells were carried out as we previously described [3]. Ovarian cancer tissues belonging to different histotype (OV-1, serous adenocarcinoma; OV-2, embryo sinus carcinoma; OV-3, cyst-myxomatous adenoma) were freshly obtained from Peking Union Hospital with informed consent provided by the patients. The use of human tissues for research was also approved by the ethics committee of the Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, Guangzhou Institute of Pediatrics (2015020917).

Gene sequencing of hmsh2 in HO8910 cells

Appropriate numbers of HO8910 cells in the logarithmic growth phase were collected for total mRNA extraction and cDNA reverse transcription. The target hmsh2 gene was amplified using fragment PCR as we described in Subcellular hMSH2 expression is aberrant in membrane-hMSH2-overexpressing cervical, lung and gastric cancer cell lines and tissues (article in progression). Three PCR products with predicted lengths (990, 1250 and 1549bp) were purified and introduced into the pGEM-T easy vector (Promega, USA). Recombinant plasmids were positively selected and characterized by Not I digestion before gene sequencing. Human msh2 variants in HO8910 cells were screened by alignment with the full-length hmsh2 sequence (NM_000251) using Laser gene 7 software.

Soluble full-length and truncated forms of hMSH2 in the whole cell extracts and culture supernatant of HO8910 cells

Soluble forms of hMSH2 protein in the complete protein extracts and the cell culture supernatant of HO8910 cells were simultaneously separated by SDS-PAGE and blotted with antihMSH2 McAb (65021-1 Ig, 1:500, Proteintech Group, USA) and with goat anti-mouse IgG/HRP secondary antibody (1:5000, Zhongshan Jinqiao Company, China). 𝛽-actin was blotted as endogenous reference control (MW 43 kDa).

Growth curves of HO8910 cells

For growth curves of both OSC cell lines, appropriate numbers of trypsin-digested HO8910, SKOV3 and HK-2 cells were planted in 24-well plates in triplicate and cultured for 8 consecutive days in a 5% CO2 atmosphere at 37°C. Growth curves of the examined cell lines were obtained by using the cell counting method. The expansion rates of target OSC cells were compared with that of HK-2 control cells.

Quantitative real-time PCR for hmsh2 mRNA expression in HO8910 cells

The human msh2 gene transcription levels in HO8910, SKOV3 and HK-2 cells were comparatively analyzed by qRT-PCR. The results were processed with Sequence Detector Version 1.2 (Applied Biosystems, USA) and Sigma Plot 11.0 as we previously described [3].

Ectopic membrane, cytoplasmic and nuclear expression of hMSH2 in HO8910 cells

The membrane, cytoplasmic and nuclear hMSH2 expression in HO8910, SKOV3 and HK-2 cells were further investigated by laser confocal microscopy with different fixation reagents. The target cells (2-3×105) were cultured overnight on pre-autoclaved glass cover slips in a 24-well format, properly fixed with 4% paraformaldehyde (PFA) or ice-cold methanol for 10-15 min and blocked with 0.5% BSA for 30 min at 4°C before labeling with specific anti-hMSH2 (N- 20, Santa Cruz Biotechnology, USA) polyclonal antibody or rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated goat antirabbit secondary antibody (Zhongshan Jinqiao Company, Beijing, China). The nuclei of the examined tumor cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) (1:1000, Sigma, USA) before being mounted on a Leica DMIRE2 inverted microscope (objective, 40×; numerical aperture, 1.25).

Participation of mhMSH2/TCRγδ/NKG2D in Vγ9Vδ2 T cell-mediated anti-OSC immunity

For further analysis of the role of mhMSH2 in human Vγ9Vδ2 T cell-mediated anti-OSC immunity, effector Vγ9Vδ2 T cells were incubated with SKOV3 cells at different ratios (effector: target [E: T] 20:1, 10:1, 5:1 and 2.5:1). Cytotoxicity was measured with the LDH method, as we previously described [3]. For cytotoxicity blockade assays, target OSC cells were pretreated with anti-hMSH2 (N20, Santa Cruz Biotechnology), anti-NKG2D (149810, R&D Systems) and anti-TCRγδ (B1.1, Immunotech, France) antibodies before incubation with effector Vγ9Vδ2 T cells at different E:T ratios (20:1, 10:1, 5:1 and 2.5:1). The blockade cytotoxicity was measured and compared to that of the rabbit IgG blockade control group. Target SKOV3 cells were stained with anti-hMSH2, anti-hMSH3 and anti-hMSH6 antibodies to confirm the cell surface expression of hMSH2/3/6 antigens before cytotoxicity and antibody-blocking cytotoxicity assays.

Subcellular hMSH2/3/6 Distribution In OSC Tissues

For IHC analyses, freshly collected ovarian cancer tissues were classically made into paraffin sections (3-4 μm in thickness) after proper neutral formalin fixation. The biopsies were heated at 60°C overnight and gradient dehydrated with methanol before antigen retrieval in boiled citrate buffer, followed by overnight incubation with purified mouse anti-hMSH2 monoclonal antibody (mAb) (clone G219-1129,1:200), mouse anti-hMSH3 mAb (clone 52, 1:20), mouse anti-hMSH6 mAb (clone 44, 1:30) (BD Pharmingen, USA) or isotype-matched mIgG1 at 4°C in a moist environment after 3% H2O2 treatment. The coloration was developed with PV9000 reagents (Zhongshan Jinqiao Company, Beijing, China) and captured with a Leica DM3000 imaging system.

Statistical Analysis

GraphPad Prism 7.2 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis and data plotting. Data are presented as the mean ± SD. Differences between/among groups were compared with Student’s t-test (two-tailed) or one-way ANOVA, P<0.05 was considered statistically significant.

Results

Hmsh2 gene sequencing and soluble full-length and truncated hMSH2 protein blotting

 

The PCR products of hmsh2 in HO8910 cells were 990, 1250 and 1549 bp in length (Figure 1A). By aligning the spliced full gene sequence with hmsh2 (NM_000251), we observed a point mutation (hmsh2 c.2332 T>A) in the HO8910 cell line (Figure 1B). It was a silent mutation that did not alter the primary structure of hMSH2 protein. The gene sequencing data of mutated hmsh2 in HO8910 cells were submitted to GenBank (Accession number: MG674653) for release. No additional gene mutation of hmsh2 was found in other mhMSH2-overexpressing epithelial tumor cell lines (data not shown here). By SDS-PAGE separation and western blots, several soluble forms of hMSH2 proteins were identified from the whole HO8910 cell protein extract [including the full-length form (MW ~105 kDa) and four truncated variants (MW~70, ~62, ~55 and ~40 kDa)] and the cell culture supernatant (MW~88, ~68 kDa) (Figure 1C).

Hmsh2 gene transcription and ectopic cytoplasm/ membrane expression

The growth and proliferation of both OSC cell lines were depicted with growth curves determined by cell counting assays. Both HO8910 cells and SKOV3 cells achieved rapid growth on day 5 and expanded much faster than the normal control HK-2 cells (Figure 2A). The mRNA expression of hmsh2 was slightly decreased in SKOV3 cells but substantially elevated in HO8910 cells compared with HK-2 cells (Figure 2B). Further detection of the ectopic membrane overexpression of hMSH2 on target OSC cells with laser confocal microscopy showed that both HO8910 and SKOV3 displayed different degrees of green fluorescence on the cell surface after labelling with a specific anti-hMSH2 antibody. The cell surface green fluorescence was much stronger on SKOV3 cells than on HO8910 cells. No green fluorescence was observed on normal control HK-2 cells (Figure 2C, the upper panels of pictures). The subcellular distribution of hMSH2 in HO8910 and SKOV3 cells was further determined by confocal microscopy with ice-cold methanol fixation. The membranes of both cells displayed different degrees of green fluorescence. The density of green fluorescence in the cytoplasm and the nuclei of SKOV3 cells was much stronger than that of HO8910 cells. There was no green fluorescence observed in HK-2 cells (Figure 2C, the lower panels of pictures).

 

Previously, we identified the membrane-overexpressed hMSH2 on several epithelial tumor cell lines as a stress-inducible endogenous protein ligand for human Vγ9Vδ2 T cells [3,6]. Herein, mhMSH2-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells towards target OSC cells were confirmed by cytotoxicity and independent specific antibody blockade cytotoxicity assays. mhMSH2 overexpression on OSC targets was validated with flow cytometry (FCM) before cytotoxicity and cytotoxicity blockade assays (Figure 3A). At E:T ratios of 20:1, 10:1, 5:1 and 2.5:1, the cytotoxic efficiency of effector human Vγ9Vδ2 T cells against target SKOV3 cells was 53%, 26%, 23% and 12%, respectively (Figure 3B). The ectopic mhMSH2-mediated recognition and cytotoxicity could be strongly blocked by specific anti-hMSH2, anti-NKG2D or anti-TCRγδ antibodies at different E:T ratios, indicating the participation of mhMSH2 in Vγ9Vδ2 T cell-mediated anti-OSC immunity by NKG2D/γδ TCR recognition (Figure 3C).

 

Subcellular distribution of hMSH2/3/6 in OSC tissues

As shown in Table 1, all ovarian cancer cells displayed positive ectopic cytoplasmic and/or membrane hMSH2/6 expression and cytoplasmic and/or nuclear hMSH3 expression, while loss of nuclear expression of hMSH2 was observed in all 3 categories of ovarian cancer tissues. Strong and clear cytoplasmic and/or membrane hMSH2/3/6 expression was strikingly observed in ovary cyst-myxomatous adenoma nests (Figure 4A). Total loss of nuclear expression of hMSH6 was found in 66.67% (2/3) of the examined ovarian cancer tissues (Figure 4A). hMSH2/3/6 expression showed substantial heterogeneity among individual ovarian cancer patients (Figure 4B).

 

Discussion

Human msh2 is one of the most crucial genes involved in the DNA MMR pathway and was strongly associated with increased tumor mutational burden in a multivariate analysis [11]. Recent studies have shown that high hMSH2 expression is significantly associated with smoking, while low hMSH2 expression is an indicator of MMR deficiency in lung adenocarcinoma [11]. The high expression of hMSH2 accompanied by increased PD-L1 expression and CD8+ T cell infiltration therefore leads to the development of a prominent immunotherapy-responsive microenvironment for lung adenocarcinoma and acts as a potential surrogate biomarker of tumor mutational burden to identify immune checkpoint blockade responders in this disease [11]. Moreover, recent studies revealed that MSH2-MSH6 played a crucial role in activation-induced deaminase-initiated antibody diversity by recognizing uracil(s) in the Ig gene loci to generate DNA breaks [12]. Many studies have identified mutations in hmsh2 as diagnostic and/or prognostic factors in carcinomas not only in the US and Canada but also in the Middle East and China [13-15]. Human msh gene mutations also have strong potential as novel candidate triple-negative breast cancer predisposition genes and are closely associated with acute adverse events and survival in rectal cancer patients receiving postoperative chemoradiotherapy [16-19]. Pathogenic or likely pathogenic germline mutations in pms2, msh2 or msh6 were detected in 0.5% (6/1,179) of lung cancer patients [18]. In this study, we screened a novel mutation in the gene sequence of hmsh2 at c.2332 T>A in the OSC cell line HO8910. This is a silent mutation that theoretically does not result in a change to the amino acid sequence of hMSH2 protein or to the phenotype of HO8910 cells. However, a rapid expansion and a strong increase in hmsh2 mRNA expression were observed in this cell line compared with the control cell line.
Moreover, several soluble full-length or truncated variants of hMSH2 proteins were observed in the whole cell protein extract and the culture supernatant of HO8910. We deduce that these altered biological characteristics of HO8910 cells and the genesis of OSC might be linked to the hmsh2 c.2332 T>A variation, as evidence showed that germline mutations of hmsh2 and its family members were closely associated with the pathogenesis of Lynch syndrome (LS). For example, hmsh2 c.2152 C>T alteration has been recently reported as a founder mutation in Portugal; its high proportion implies combined screening for this mutation and some other previously reported founder mutations will be helpful in the genetic testing of Portuguese families with suspected LS [19]. The occurrence and clinical significance of hmsh2 c.2332 T>A and soluble full-length and truncated forms of hMSH2 proteins in clinical OSC patients remain to be clarified in the future.

Compared with HO8910 cells, SKOV3 cells displayed stronger ectopic membrane and nuclear expression of hMSH2 in laser confocal microscopy and FCM analyses, suggesting better membrane anchoring and a more efficient nuclear importing system in SKOV3 cells. The ability of the notably overexpressed mhMSH2 on SKOV3 cells to promote human Vγ9Vδ2 T cell-mediated recognition and cytotoxicity via TCRγδ/NKG2D receptors towards target OSC cells was later validated by independent cytotoxicity and specific antibody blockade cytotoxicity assays, providing further evidence for mhMSH2 functioning as an OSC-associated self-antigen (ligand) for human Vγ9Vδ2 T cells in anti-ovarian cancer immunity [10]. In the absence of information/data demonstrating that the observed mutation impacts function, one cannot determine if the mutation is physiologically relevant. In further investigations on the subcellular distribution of hMSH2 and its companion proteins in ovarian cancer tissues, we found that all examined ovarian cancer specimens (serous adenocarcinoma, embryo sinus carcinoma, cystmyxomatous adenoma) displayed positive ectopic cytoplasmic and/or membrane hMSH2/6 expression and cytoplasmic and/or nuclear hMSH3 expression, and total loss of nuclear expression of hMSH2/6 commonly occurred in almost all of the ovarian cancer tissues. Considering the high hmsh2 gene transcription and the loss of nuclear distribution of hMSH2 protein in HO8910 cells, we hypothesized that the nuclear importing system of hMSH2 and/ or hMSH6 protein was dysfunctional. By contrast, recently, it has been reported that a high overall rate (16.2%) of MMR deficiency was surprisingly observed in ovarian endometrioid carcinoma and was significantly associated with increased IFOG (International Federation of Obstetrics and Gynecology) grade and CD8+ intraepithelial lymphocyte infiltration but not with cancer-specific death [19]. These findings suggest a promising future in which loss of nuclear expression of hMSH2 and/or hMSH6 protein may function as effective screening/diagnostic markers or therapeutic targets for different subtypes of ovarian cancers and as endogenous immune ligands not only for Vγ9Vδ2 T cells [20]. We expected that a human cell-based assay system for functional testing of hMSH2 and its family members will facilitate the identification of highrisk ovarian carcinoma patients and the generation of individual autoantigen-targeted immunotherapies for OSC.

Conclusion

In this study, we identified a silent hmsh2 c.2332 T>A mutation (GenBank accession no. MG674653) along with serval novel soluble truncated forms of hMSH2 variants in HO8910 cells and validated the mhMSH2/TCRγδ/NKG2D-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells against mhMSH2/6-overexpressing SKOV3 cells. We also demonstrated the abnormal subcellular distribution of hMSH2/3/6 in the examined ovarian cancer tissues. The clinical significance of the critical findings in OSC genesis and human Vγ9Vδ2 T cell-mediated anti-OSC immunity remain to be further investigated.

 

https://lupinepublishers.com/pharmacology-clinical-research-journal/fulltext/a-hmsh2-c2332-ta-mutation-and-membrane-hmsh2-tcr%CE%B3%CE%B4-nkg2d-mediated-cytotoxicity-of-human-v%CE%B39v%CE%B42-t-cells-in-ovarian-serous-cystadenocarcinoma.ID.000142.php

https://lupinepublishers.com/pharmacology-clinical-research-journal/pdf/LOJPCR.MS.ID.000142.pdf

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