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ORIGINAL ARTICLE
Ahead of print publication  

The impact of Momordica charantia treatment on cisplatin-induced adverse effects in albino rats


1 Department of Biology, Faculty of Sciences, King Khalid University, Abha, Kingdom of Saudi Arabia; Department of Zoology, Faculty of Sciences, Mansoura University, Mansoura, Egypt
2 Department of Biology, Faculty of Sciences, King Khalid University, Abha, Kingdom of Saudi Arabia
3 Department of Pathology, Faculty of Medicine, King Khalid University, Abha, Kingdom of Saudi Arabia; Department of Pathology, National Liver Institute, Menoufia University, Al Minufiyah, Egypt

Date of Submission04-Jan-2020
Date of Decision27-May-2020
Date of Acceptance16-Jul-2020
Date of Web Publication21-Jan-2021

Correspondence Address:
Fatma Mohsen Shalaby,
King Khalid University, Faculty of Sciences, Biology Department, Abha

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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_18_20

 > Abstract 


One of the major antineoplastic drugs is cisplatin that has a dose-dependent toxicity. Momordica charantia (bitter melon), a natural healthy vegetable, and its metabolites possess hypoglycemic, antioxidant, anticarcinogenic, and other beneficial properties.
Aim: This study evaluates the effect of combined cisplatin and bitter melon extract (BME) treatment on liver and kidney of male albino rats.
Subjects and Methods: The animals were subjected to intraperitoneal injection of cisplatin (1 mg/kg body weight) and concurrent oral treatment of BME, 300 mg/kg body weight. The effect of cisplatin/BME co-treatment on liver and kidney was determined by evaluating the histopathological changes and immunohistochemical expression of apoptotic markers: caspase-3, P53, and Bcl-2 and PCNA as a proliferative indicator.
Results: This study shows that cisplatin/BME co-treatment improves cisplatin-induced hepatic but not renal pathological changes. It decreases significantly the proliferative activity in liver and renal tissues and augments some apoptotic pathways in both organs.
Conclusion: Administration BME alone did not show any undesirable side effects on hepatic and renal biochemical or pathological levels as cisplatin. It enhances apoptosis and inhibits proliferation on molecular levels. Combined cisplatin/BME treatment shows more apoptotic and antiproliferative effects and enhances nephrotoxicity. Therefore, concurrent consumption of BME and cisplatin is not advisable in vivo. The antiproliferative potential of BME renders it a possible alternative option for cancer therapy taking into consideration the dose and duration of treatment. Further, in vivo studies are needed to investigate whether administration of specific BM ingredients alternative or consecutive with cisplatin may enhance its apoptotic and antiproliferative efficacy.

Keywords: Cisplatin, histopathology, immunohistochemical staining, Momordica charantia



How to cite this URL:
Shalaby FM, Alshehri MA, Elrefaie AO. The impact of Momordica charantia treatment on cisplatin-induced adverse effects in albino rats. J Can Res Ther [Epub ahead of print] [cited 2021 Jun 24]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=307514




 > Introduction Top


Cisplatin (cis-diamminedichloroplatinum) is an effective cancer chemotherapy that plays a significant role in the treatment of many malignancies.[1] It disrupts nuclear and mitochondrial DNA, suppresses DNA replication, enhances the formation of reactive oxygen species (ROS), and induces apoptosis.[2] ROS perform a significant role in mediating programmed cell death by activation of caspases, decreasing antioxidant capacity, and increasing mitochondria and DNA damage.[3] Various studies confirmed that treatment with cisplatin is associated with numerous, cumulative, and dose-dependent adverse effects.[4] It may induce nephrotoxicity, ototoxicity, hepatotoxicity, cardiotoxicity, and neurotoxicity[3] resulting in many biochemical and histological alterations.[4] The kidney is the most affected organ as it is the main route of cisplatin excretion.[2] Cisplatin combination chemotherapy is the basis of treatment of many cancers.[3]

Administration of antioxidants may prevent or ameliorate the toxic effects of cisplatin.[5]

Momordica charantia, commonly known as karela or bitter melon (BM), is used as a popular medicine in many countries. Bitter melon extract (BME) has potent antioxidant and free radical scavenging properties attributed to compounds such as flavonoids and phenols.[6] The antitumor activity of BM has begun to attract attention; for instance, the plant crude extract was used in treatment of adrenocortical,[7] breast,[8] and prostate[9] cancer cell lines. In vitro studies showed that BME suppressed cancer cell proliferation by modulating the regulatory genes of cell cycle and enhancing apoptosis.[8]

Although there are some reports in the literature about the enhancement effect of BME on the antiproliferative property for chemotherapeutic agents such as cisplatin,[10] the effects and pathogenesis of cisplatin/BME co-treatment in normal tissues in vivo are still not fully illustrated. Thus, the aim of the current study is to investigate the efficacy of BME in ameliorating the hepatic and renal adverse effects of cisplatin and to evaluate whether it has an adjuvant role in increasing the apoptotic and antiproliferative potentials of cisplatin in albino rats.


 > Subjects and Methods Top


Chemicals

Cisplatin

Cis-diamminedichloroplatinum (II) powder dissolved in saline (1 mg/ml) was obtained from Sigma-Aldrich (Germany).

Bitter melon extract preparation

Fresh bitter melon fruits (M. charantia) were bought from commercial local markets in Abha city, Saudi Arabia. The fruits were identified by a botanist, College of Science, KKU, Saudi Arabia. The aqueous plant extract was prepared according to Nawwar et al.[11] with slight modification as follows: the fruits were washed with distilled water. The fleshy parts were cut into small pieces, air-dried, and crushed into fine powder. The powder was dissolved in distilled water and left in water bath (Fisher Scientific-2232, USA) for 1 h at 60°C and then overnight at room temperature. The extract was filtered and concentrated using a vacuum rotatory evaporator system (HB-10, IKA) at a constant temperature of 45°C and then dried in an oven. The dried extract was stocked at 20°C till use.

Animals

Male albino rats weighing 150–200 g were used. They were housed in plastic cages in an air-conditioned animal house at 25°C ± 5°C with specific pathogen-free conditions, Department of Biology, Faculty of Sciences, KKU, Saudi Arabia. They were subjected to a 12/12-h daylight/darkness and allowed unlimited access to water and food. Prior permission for animal use and housing and approval of the protocol (Approval No. ECM#2020-0708) were obtained from the Institutional Animal Ethical Committee, Faculty of Sciences, KKU, Saudi Arabia.

Experimental design

The animals were divided into four groups, six animals each. Group 1: The rats received intraperitoneal (ip) injection of normal saline and served as a control group. Group 2: The rats received oral BME 300 mg/kg through gastric tube day after day for 3 weeks.[12] Group 3: The rats received ip injection of cisplatin (1 mg/kg) once day after day for 21 days.[13] Group 4: The rats received cisplatin in a dose similar to Group 3 and oral BME 300 mg/kg for 3 weeks. At the end of the experiment (3 weeks), the rats were fasted overnight and then humanely killed. Blood was drawn through heart puncture into tubes, and the livers and kidneys were removed. Serum was separated by centrifuging at 3000 rpm for 15 min at room temperature, collected carefully, and kept at −20°C until analysis. The tissue specimens of liver and kidneys were immediately fixed and processed for histopathological and immunohistochemical investigations.

Biochemical analysis

Spectrophotometer is used to estimate serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels using commercial kits according to Reitman and Frankel[14] and creatinine and blood urea nitrogen (BUN) according to Lamb et al.[15]

Histological examination

Slides were coded, stained with H and E stain, and then examined.

Immunohistochemical staining for caspace-3, Bcl-2, P53, and PCNA

Liver and kidney sections (4 μm) thick were mounted on glass slides (poly-L-lysine-coated) and stained immunohistochemically for caspase-3, Bcl-2, and P53 expression to assess apoptosis and for PCNA as a proliferative marker. Immunostaining was performed with standard streptavidin-biotin immunoperoxidase method (LSAB kit; Dako Japan Corp., Kyoto, Japan). Sections were deparaffinized in xylene and rehydrated through gradual alcohol series. For antigen retrieval, the sections were heated in a microwave oven (medium-to-low temperature) for 20 min in 0.1 mol/L sodium citrate buffer (pH 6.0). To bleach endogenous peroxidase activity, the sections were immersed in 3% hydrogen peroxide in methanol for 30 min followed by rinsing 3 times in Tris buffer (pH 7.4) for 10 min. Ultra V Block was applied for 5 min at room temperature to reduce nonspecific background staining. To detect caspace-3, Bcl-2, P53, and PCNA immunoreactivities, sections were incubated in a humid chamber with the following: rabbit polyclonal anti-activated caspase-3 (1:400; Thermo Fisher Scientific, USA), rabbit polyclonal anti-Bcl-2 (1:400; Thermo Fisher Scientific, USA), mouse monoclonal anti-P53 (1:100; Genemed, USA), and mouse monoclonal PCNA antibody (1:200; Thermo Fisher Scientific, USA) overnight at 4°C. Then, the slides were washed 3 times in Tris buffer for 10 min each. Rabbit anti-mouse antibody was used as the linker molecule for 20 min. After washing by Tris buffer, tissues were visualized with 3,3'-diaminobenzidine and counterstained with hematoxylin. Finally, the sections were dehydrated in xylene, mounted with DPX, coverslipped, and examined. Positive and negative controls were conducted in parallel for each antibody to test their specificity.

Image analysis

Olympus digital camera on an Olympus microscope with 0.5X photo adaptor was used for photographing slides. The images were analyzed on Intel (Core I3)-based computer using VideoTest-Morphology Software VideoTesT (Saint Petersburg, Russian Federation) with a specific built-in routine for object counting and analysis. Five random fields in five slides for each group were analyzed. Images were threshold at the level of the desired hue range (positive and negative stains) to form a binary mask that represents target objects. Then, binary masks were identified as region of interest (ROI), and then, object counting routine was applied on both ROIs (with area and circularity exclusion filter), from which % positive cells were calculated. All results were exported as XLS for statistical analysis.

Statistic analysis

Data were analyzed using SPSS software computer program version 23 for Microsoft Windows (SPSS Inc software., Chicago, IL, USA). Data were quantitative parametric and expressed as mean and standard deviation. One-way analysis of variance followed by post hoc Tukey was used for comparing quantitative parametric data. P < 0.05 was considered as statistically significant.


 > Results Top



 > Biochemical results Top


Administration of BME resulted in no significant changes in kidney or in liver function tests. Hepatotoxicity and nephrotoxicity were evident by impairment of hepatic and renal functions in Group 3. Rats in Group 3 showed a significant increase in serum levels of ALT, AST, and creatinine comparable to Group 1 and 2. The increase in BUN did not reach a significant value. Group 4 exhibited a nonsignificant decrease in ALT, AST, creatinine, and BUN than that of Group 3 [Table 1].
Table 1: Effects of bitter melon extract, cisplatin, and bitter melon extract/cisplatin co-treatment on serum aspartate aminotransferase and alanine aminotransferase, creatinine, and blood urea nitrogen in rats (mean±standard deviation)

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Histological examination

Liver sections of Group 1 (control) showed normal liver parenchyma and portal areas [Figure 1]a. In Group 2 (BME-treated rats), the central veins and portal vein branches were congested [Figure 1]b. Cisplatin treatment (Group 3) resulted in more sinusoidal and portal vein congestion, moderate portal mononuclear cell infiltration, spotty necrosis, many hepatocytes with pyknotic nuclei and deep eosinophilic cytoplasm (apoptotic cells), Kupffer cell hyperplasia, and disorganization of the hepatic cords [Figure 1]c and [Figure 1]d. Well-formed granulomas were also seen [Figure 1]e. Cisplatin/BME co-treatment (Group 4) improved cisplatin-induced inflammatory and necrotic hepatic lesions; only ballooning of hepatocytes was seen [Figure 1]f. Sections of rat kidneys showed normal glomeruli, tubules, blood vessels, and interstitial tissue with no relevant pathological changes in Groups 1 and 2 [Figure 2]a and [Figure 2]b. In Group 3, diffuse distortion of glomerular tufts with widening of Bowman's spaces, vacuolization, segmental dilation and atrophy of renal tubules, and interstitial inflammatory infiltrate were found [Figure 2]c. Proximal convoluted tubules were lined by cells having deeply eosinophilic cytoplasm and pyknotic nuclei (apoptotic cells) that detached from tubular basement membrane. Other tubular cells showed disruption of their luminal borders. Many renal tubules contained cellular and hyaline casts [Figure 2]d. No histological improvement of the renal glomerular, tubular, and interstitial tissue pathology was found in Group 4 comparable to Group 3 [Figure 2]e and [Figure 2]f.
Figure 1: Liver histology (H and E): (a) Control untreated rats: normal liver tissue, ×100. (b) BME-treated rats: congested central veins (*) and portal vein branch, ×100. Cisplatin-treated rats (c-e): (c) sinusoidal (*) and portal vein branch (arrowhead) congestion, and moderate portal mononuclear cell infiltration (arrow), ×200. (d) Spotty necrosis (arrowhead), disorganization of the hepatic cords (*), hepatocytes with pyknotic nuclei (arrow), and Kupffer cell hyperplasia × 200. (e) A well-formed granuloma (arrowhead) and many pyknotic nuclei (arrows) ×200. (f) Cisplatin and BME co-treated rats. Improvement of all pathological changes, however, marked ballooning of hepatocytes is seen × 100

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Figure 2: Kidney histology (H and E): (a) Control untreated rats: normal glomeruli and tubules × 200. (b) BME-treated rats: no pathological changes × 200. (c-d) Cisplatin-treated rats, (c) diffuse distortion of glomerular tufts with widening of Bowman's spaces (*) vacuolization, segmental dilation and atrophy of renal tubules (arrowheads), interstitial inflammatory infiltrate (arrow), ×200. (d) Proximal convoluted tubules with deeply eosinophilic cytoplasm and pyknotic nuclei (arrow), detachment of tubular epithelial cells, disruption of the luminal borders of renal tubular epithelial cells (arrowheads), intraluminal cellular debris and hyaline casts (*) ×200. (e-f) Cisplatin and BME co-treated rats, (e and f, ×100) no histological improvement of the glomerular and tubular pathology comparable to cisplatin group

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Immunohistochemical staining

For caspase-3

In Group 1, weak cytoplasmic positive signals for caspase-3 were seen in individual liver cells [Figure 3]a and in renal tubular epithelium [Figure 4]a. Image analysis showed that administration of either BME (Group 2) or cisplatin (Group 3) resulted in a significant increase of caspase-3 expression comparable to Group 1 in liver [Figure 3]b and [Figure 3]c and in kidney [Figure 4]b and [Figure 4]c tissues. It is worth noting here that caspase expression was significantly higher in liver tissue of Group 3 than in Group 2. BME/cisplatin co-treatment (Group 4) resulted in a significant intensive expression of caspase-3 in liver [Figure 3]d and kidney tissues [Figure 4]d comparable to Group 2 and 3 [Table 2] and [Table 3].
Figure 3: Liver IHC-caspase-3: (a) Control untreated rats: no/very weak cytoplasmic staining in individual hepatocyte. (b) BME-treated rats: moderate cytoplasmic and membranous hepatocellular staining. (c) Cisplatin-treated rats: moderate cytoplasmic and membranous staining of many hepatocytes. (d) Cisplatin and BME co-treated rats: intense cytoplasmic and membranous staining of many hepatocytes (×400)

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Figure 4: Kidney IHC-caspase-3: (a) Control untreated rats: very weak cytoplasmic staining of tubular epithelium. (b) BME-treated rats: moderate tubular cytoplasmic staining. (c) Cisplatin-treated rats: intense tubular cytoplasmic staining. (d) Cisplatin and BME co-treated rats: more intense positive staining (×400)

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Table 2: Caspase-3, P53, Bcl-2, and proliferating cell nuclear antigen immunostaining in rat livers (mean±standard deviation)

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Table 3: Caspase-3, P53, Bcl-2, and proliferating cell nuclear antigen immunostaining in rat kidneys (mean±standard deviation)

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For P53

In Group 1, weak cytoplasmic positive signals for P53 were seen in individual liver cells [Figure 5]a and in renal tubules [Figure 6]a. P53 protein expression increased significantly in the liver and kidney tissues of Groups 2, 3, and 4 than in Group1 with no statistically significant difference between each group and the others [Figure 5], [Figure 6]a, [Figure 6]b, [Figure 6]c, [Figure 6]d and [Table 2], [Table 3].
Figure 5: Liver IHC-P53: (a) Control untreated rats: weak cytoplasmic staining in individual hepatocyte. (b) BME-treated rats, (c) Cisplatin-treated rats, and (d) Cisplatin and BME co-treated rats: moderate cytoplasmic staining of hepatocytes with no significant difference between the three groups (×400)

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Figure 6: Kidney IHC-P53: (a) Control untreated rats: weak cytoplasmic staining of tubular epithelium. (b) BME-treated rats, (c) Cisplatin-treated rats, and (d) Cisplatin and BME co-treated rats: moderate cytoplasmic staining of renal tubules with no significant difference between the three groups (×400)

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For Bcl-2

In Group 1, the liver and kidney sections showed a moderate-to-strong cytoplasmic and membranous staining for Bcl-2 in individual hepatocyte and Kupffer cells and in renal glomeruli and tubules. Bcl-2 expression in the liver and kidney tissues decreased significantly to almost 50% in Group 2 comparable to Group 1 [Figure 7], [Figure 8]a, [Figure 8]b and [Table 2], [Table 3]. It showed marked significant attenuation in Group 3 comparable to Groups 1 and 2 [Figure 7] and [Figure 8]c. BME/cisplatin co-treatment resulted in a significant improvement in Bcl-2 expression [Figure 7] and [Figure 8]d comparable to Group 3 but not to Group 2. Bcl-2 expression in Group 4 is significantly less than in Group 1 [Figure 7], [Figure 8]a, [Figure 8]b, [Figure 8]c, [Figure 8]d and [Table 2], [Table 3].
Figure 7: Liver IHC-Bcl-2: (a) Control untreated rats: moderate hepatocellular cytoplasmic and membranous staining. (b) BME-treated rats: weak cytoplasmic and membranous staining of hepatocytes. (c) Cisplatin-treated rats: no positive staining. (d) Cisplatin and BME co-treated rats: moderate cytoplasmic staining in occasional hepatocytes (×400)

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Figure 8: Kidney IHC-Bcl-2: (a) Control untreated rats: very intense cytoplasmic staining of tubular epithelium. (b) BME-treated rats: moderate tubular cytoplasmic staining. (c) Cisplatin-treated rats: weak tubular cytoplasmic staining. (d) Cisplatin and BME treatment reduces the intensity of staining (×400)

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For PCNA

Liver and kidney PCNA staining showed diffuse nuclear staining in hepatocytes and renal tubular epithelium. In Groups 2, 3, and 4, there was a significant decrease in liver PCNA protein expression comparable to Group 1with no significant difference between them [Figure 9]a, [Figure 9]b, [Figure 9]c, [Figure 9]d and [Table 2]. A reduction of PCNA expression in renal tissue of Groups 2, 3, and 4 comparable to Group 1 was seen. The difference was not significant between Groups 1 and 2 and reached a significant level between Groups 3 and 4 comparable to Group 1. In Group 4, the attenuation in PCNA expression was significantly comparable to Group 2 but not to that of Group 3 [Figure 10]a, [Figure 9]b, [Figure 9]c, [Figure 9]d and [Table 3].
Figure 9: Liver IHC-PCNA: (a) Control untreated rats: positive nuclear staining in many hepatocytes. (b) BME-treated rats: weak nuclear staining. (c) Cisplatin-treated rats and (d) cisplatin and BME co-treated rats: Very weak or no staining (×200)

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Figure 10: Kidney IHC-PCNA: (A) Control untreated rats: very few positive nuclei. (B) BME-treated rats and (C) Cisplatin-treated rats and (D) Cisplatin and BME co-treated rats: no staining (×400)

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 > Discussion Top


Cisplatin is one of the most widely used anticancer drug, however, it may cause hepatotoxicity and nephrotoxicity that depend on the dose and the duration of the drug administration.[3] In the present study, BME is used in combination with cisplatin as an antioxidant and anti-inflammatory agent to ameliorate cisplatin-induced toxicity.

In this study, cisplatin administration led to deteriorative biochemical and histopathological changes in liver and kidney of rats. Hepatotoxicity and nephrotoxicity were evident by a significant increase of ALT, AST, and serum creatinine levels. Our data were in agreement with Palipoch and Punsawad[4] who observed a significant increase in serum levels of ALT and AST, creatinine, and BUN in cisplatin-treated rats. Histopathological changes in the liver run parallel with many studies and showed that cisplatin induced liver tissue injury; degeneration, apoptosis and necrosis of hepatocytes, portal inflammatory cell infiltrate and sinusoidal dilatation.[16],[17] The pathological alterations were more obvious in the kidney tissue, particularly in renal tubules that showed vacuolization, apoptosis, detachment of tubular epithelium, and tubular casts. In addition, the glomeruli were distorted and the interstitial tissue was infiltrated by mononuclear cells.[18] The detrimental effect of cisplatin on kidney is attributed to the fact that kidneys are the main route of cisplatin excretion where its concentration in proximal tubular cells is about fivefold serum concentration.[19] It enters renal cells by passive and/or facilitated mechanisms where it is biotransformed into a more potent toxin through formation of glutathione conjugates.[20] In addition, cisplatin induces TNF-α production in tubular cells, which triggers a robust inflammatory response, further contributing to tubular cell injury and death. Renal vasculature injury that leads to ischemic tubular cell death and decreased glomerular filtration rate is another mechanism by which cisplatin may induce renal damage.[2],[3] The cisplatin-associated alterations in liver and kidney whether biochemical or pathological are attributed to the induction of cellular oxidative stress and endoplasmic reticulum stress that increase the level of superoxide anions and hydroxyl radicals.[21] These free radicals can directly react with different cellular components causing their damage, inhibit mitochondrial respiratory chain functions, and increase mitochondrial permeability transition pore leading to ATP depletion and nucleus-independent apoptosis signaling.[3] In addition, cisplatin leads to direct necrosis and apoptosis mediated by activation of cell death-promoting signaling pathways: mitogen-activated protein kinases, p53, and ROS.[3] Several studies have suggested that adjuvant therapy for cisplatin can reduce the undesirable side effects and overcome drug resistance.[10],[22],[23] It has been shown that M. charantia extracts have excellent free radical scavenging and anti-inflammatory properties. It inhibits stress-induced lipid peroxidation by its several biologically active constituents, such as flavonoids and phenols.[24]

In the current study, oral administration of BME in a dose 300 mg/kg day after day for 21 days did not result in any biochemical or histopathological changes in rat liver or kidney; only mild hepatic venous congestion was observed. Our data run parallel with Thenmozhi and Subramanian[12] who showed that oral administration of 300 mg/kg of ethanolic extract of BM fruit for 8 consecutive weeks to rats did not induce any significant changes in the blood ammonia, plasma urea, and liver enzyme markers.

Furthermore, El Batran et al.[25] reported insignificant change in serum ALT and AST, urea, and creatinine after administration of three doses of BM juice or alcohol extracts. Mardani et al.[26],[27] and Houéto et al.[28] showed that a high dose up to 4000 mg/kg of ethanolic fruit BME as a single dose has no significant adverse effects on renal or liver functions or structures. However, long-term consumption of 500 mg/daily for 7 days resulted in some renal complications. Contrarily, Tennekoon et al.[29] showed that oral fruit juice and seed extract of BM administration at a daily dose of 1 ml/100 g body weight for 30 days lead to significant elevation in hepatic enzyme without significant histopathological abnormalities. They attributed their findings to the damage of hepatocyte caused by BM at the molecular level. The data from the aforementioned studies indicate that the dose and duration of BME consumption could have diverse effects on biochemical tests and histology. This means that for ensuring the safety of BM consumption, separation, purification, and identification of specific therapeutic constituents are required.

This study showed that cisplatin/BME co-treatment led to nonsignificant decrease in hepatic enzymes associated with noticeable improvements in hepatic tissue inflammation and necrosis than that of cisplatin-treated animals. Furthermore, Teoh et al.[30] reported an improvement of hepatic pathological changes in streptozotocin-induced diabetic rats after treatment with aqueous extract of BM at dose of 50 mg daily for 10 days. This improvement could be attributed to the decrease of lipid peroxidation and inhibition of overproduction of inflammatory mediators, especially pro-inflammatory cytokines IL-1b, IL-6, and TNF-α.[31] Gene expression analysis showed that bitter melon butanol fraction suppressed expression of various genes controlling the inflammatory process.[32]

Cisplatin/BME co-treatment in the present study showed no detectable improvement of renal functions or histopathological changes. Contrary, earlier studies revealed nephroprotective efficacy of BME against oxidative stress induced either by diabetes or CCl4.[31],[33] The multi-mechanisms by which cisplatin produces renal damage make the amelioration of renal pathology very difficult.[34] Cisplatin induces oxidative burst with formation of ROS, intracellular calcium overload, inhibition of mitochondrial respiratory chain function, opening of mitochondrial permeability transition pore, ATP depletion, and induction of apoptosis and necrosis. The high concentration of cisplatin and its potent toxic metabolites in renal tubules augments all aforementioned pathways.[19],[35] Moreover, the synergistic action of cisplatin/BME co-treatment adds more burden on renal tissue causing more tubular and glomerular damage.

To clarify the cellular and molecular mechanisms by which cisplatin/BME co-treatment affected the liver and kidney of male albino rats, immunohistochemical techniques were used to investigate the proapoptotic (caspase-3 and P53) and antiapoptotic (Bcl-2) protein expression and PCNA staining as a proliferative marker.

Administration of BME, cisplatin, and cisplatin/BME co-treatment in the present study resulted in almost stepwise enhancement of caspase-3 and P53 expression, especially in liver and kidney sections of cisplatin/BME co-treated rats, and downregulation of Bcl-2 expression. Previous studies on tumor cell lines revealed that BME altered the cell cycle regulatory mechanisms. It enhanced p53, p21, and pChk1/2, inhibited cyclin B1 and cyclin D1 expression, and induced apoptosis through caspases activation, alteration of Bcl-2 family expression, and cytochrome-c release into the cytosol.[8],[10],[36] P53 controls apoptosis induced by cisplatin through stimulation of proapoptotic genes to produce caspases.[37]

The normal liver and kidney histology in BME-treated rats in this study and others[38] in spite of the significant elevation of caspase-3 level may be due to the short half-life of the apoptotic figures[39] or due to exacerbation of caspase-3 that precedes the onset of apoptosis.[40] Caspase-3 is essential for certain processes associated with dismantling of the cell such as cytoskeleton damage, nuclear demise, and other apoptosis-associated cellular changes eventually to the formation of apoptotic bodies.[41] However, it may function before or at the stage when commitment to loss of cell viability is made. In addition, the activation level of caspase-3 with the used dose of BME did not reach the threshold level enough to induce cell injury.[40],[42]

It is worth mentioning here that caspase-3 expression, in the present study, was more intense in liver and kidney sections of cisplatin/BME co-treated rats, whereas PCNA expression was significantly reduced. A finding that indicates a synergistic effect of both agents in increasing the rate of apoptosis, decreasing the proliferative activity and explains the augmented renal pathological damage.[10],[40],[43] The apoptotic efficacy of BME is enhanced in the presence of cisplatin due to the state of cellular oxidative stress produced by the latter leading to activation of other caspases.[44] The increased activity of p53 in cisplatin/BME lead to the obvious renal pathological damage through mediating the induction transcription of the proapoptotic gene leading to increase the permeability of the outer mitochondrial membrane and release of apoptogenic factors. Whenever cisplatin concentration increased, the activity of caspase-3 decreased suggesting the presence of necrosis in kidney instead of apoptosis.[37] Moreover, Jiang et al.[45] reported that death of tubular epithelial cell because of cisplatin treatment is associated with activation of caspases expression either dependent or independent on P53.

Both BME and cisplatin contribute to decreased ATP production and derangement of energy homeostasis in response to external stresses. BME acts as a potential natural activator of adenosine monophosphate-activated protein kinase (AMPK), a cellular energy sensor that plays an important role in the maintenance of energy homeostasis. Many AMPK activators act as inhibitors of mitochondrial function and hence decreased ATP production.[10] In addition, BME decreases the intestinal glucose uptake and gluconeogenesis, causing hypoglycemia that leads to more activation of AMPK. Cisplatin impairs mitochondrial functions leading to further ATP depletion and AMPK activation.[46]

The significant overexpression of apoptotic signals in this study was associated with downregulation of antiapoptotic Bcl-2 expression in liver and kidney of all studied groups.[36] The initiation of apoptosis by increasing Bax/Bcl-2 ratio that subsequently leads to enhancement of mitochondria membrane permeability indicates that apoptosis induced by BME is mitochondria related.[36]

Proliferating cell nuclear antigen (PCNA) is a multifunctional protein of DNA polymerase that organizes numerous components for DNA replication, cell proliferation, cell cycle progression, and repair mechanisms.[47]

Our results showed reduced PCNA expression in BME, cisplatin, and cisplatin/BME-treated groups, indicating that either cisplatin or BME has a direct antiproliferative potential. Similarly, Hou et al.[48] reported reduced PCNA expression with cisplatin treatment. The efficacy of BME as an antiproliferative agent in the current work is consistent with the study of Ru et al.[49] who found a 51% reduction of PCNA expression in prostate tissue of BME-fed mice. It was shown that breast cancer cells treated with BME accumulated during G2-M phase of cell cycle where PCNA expression is reduced.[8] The weakest PCNA expression was found in the kidney tissue of either cisplatin or cisplatin/BME co-treated rats. It was shown that the level of PCNA expression correlates directly with rates of cellular proliferation and DNA synthesis.[34] Cell proliferation, regeneration, and fibrosis may be triggered by apoptosis.[50] Therefore, the exacerbation of apoptosis in kidney tissue is not matched by a corresponding increase in the rate of cell proliferation resulting in persistent impairment of renal functions, histological alteration, and loss of tissue/organ mass.[43]

Our results indicate that the synergistic action of both cisplatin and BME in increasing apoptosis is mediated by enhanced expression of P53 and caspase-3, and downregulation of Bcl-2 and PCNA leads eventually to cell cycle arrest, interfering cell cycle progression and proliferation. Downregulation of PCNA and increased apoptosis in BME/cisplatin co-treated rats may overcome the BME antioxidant defense mechanisms, enhance cisplatin toxicity, weaken the reparative response, and hence attenuate BME ameliorative effect of cisplatin toxicity, especially in kidney.


 > Conclusion Top


The results of the current study shed light on the therapeutic uses of bitter melon. Administration of BM fruit extract alone or combined with cisplatin enhances apoptosis and has an antiproliferative effect; however, it enhances cisplatin-induced nephrotoxicity. Therefore, concurrent consumption of BME as an ameliorative and antioxidant agent with cisplatin is not advisable in vivo. The antiproliferative potential of BME renders it a possible natural alternative option for cancer therapy. Further studies are required to verify the efficacy of specific BM antineoplastic constitutes either as an alternative or consecutive chemotherapeutic drug.

Acknowledgment

The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University, Abha, KSA, for funding this work through the General Research Project under grant number (G.R.P. 559-40).

Financial support and sponsorship

This study was financially supported by the Deanship of Scientific Research at King Khalid University, Abha, KSA, for funding this work through the General Research Project under grant number (G.R.P. 559-40).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

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