INTRODUCTION:

Ovarian cancer (OC) in women considered the fifth most popular cause of cancer death. Despite of important progress has made in treating of OC, most of patients experience recurrence of the disease and patients treated with second-line or several lines of treatment. Over the last two decades, the outcomes of primary treatment with surgery and chemotherapy, generally paclitaxel and carboplatin, have improved relatively little. However, relapse and metastasis are the primary concerns among therapists, About 80% of women in advanced OC will comprise tumor progression, or mostly a recurrence.¹

The most significant prognostic factor for ovarian cancer is the stage at which the patients present to the clinicians. A major number of ovarian cancer patients reach the clinicians at an advanced stage with peritoneal dissemination which may be due to late onset pain or other symptoms.

Interestingly the absence of clear lesions in most of the patients has resulted in a poor understanding of the metastatic mechanisms². Many biochemical markers have been identified to significantly indicate the progress of ovarian cancer³. Oxidative stress has been often interpreted contrastingly in deciding the pathophysiology of the cancer progress as well as in monitoring the effects of chemotherapy. Most of the antigenic markers like CA125 are effectively used to monitor the improvement during chemotherapy and the relapse⁴. Numerous studies have assessed the types of cancer cells to monitor the chemotherapeutic progress however the most established methods use the biochemical markers in the serum⁵. Other immunogenic markers have also been indicated to be significantly assessed for the ovarian cancer therapy⁶.

Malignant effusions, which are made up of malignant pleural and peritoneal fluid, are a rare cancer symptom that often indicates a poor prognosis⁷. Neoplastic cells originating from cancers/ tumors can migrate into the body cavities and could be threatening a metastatic outbreak. Pleural effusion is an abnormal buildup of fluid in the pleural cavity. Pleural effusion containing the malignant cells is an alarming marker for metastasized cancer. Understanding the mechanistic signals and the biochemical indices of the pleural effusions are the key to understand the parallel carcinogenetic pathways and would aid in better therapeutic strategies⁸.

The most established therapy for the ovarian cancer is cytoreductive surgical protocol followed by the platinum-based chemotherapy. The surgery will give better insight on the diagnostic confirmation and cancer staging. However, efforts should be made to define the tumor histology as well. Owing to the retrieval effects of platinum-based chemotherapy among the ovarian cancer patients, we wanted to study the effects of the first round of chemotherapy in women with ovarian cancer. The biochemical indicators may be used in deciphere the relevance between cancer retrogression and platinum recuperation.⁹

MATERIALS AND METHODS:

Participants and enrollment:

All participants (patients) were enrolled as part of clinical trial going on according to the IP and GCP/GLP guidelines as well as DGCI guidelines. Patient/ immediate relative consents were obtained for using the serum/plasma for research and data management. Forty (40) female patients having ovarian cancer were selected by the Medical Practitioner and suggested for the study (n=40). The pleural samples were collected before the initiation of chemotherapy (PEGylated cis-platin liposomes) and after three weeks of first chemotherapy session (PEG= poly ethylene glycol). Serum from non-cancer volunteers of the same age range was collected as control (n=5).

Drug and Dosage:

Pegylated liposomal cis-platin derivative is a formulation of cis-platin-derivative in polyethyleneglycol-coated liposomes with a prolonged circulation time. Cisplatin binds to base pairs in the DNA helix, limiting DNA replication and, as a result, reducing protein synthesis. Dosage is calculated by estimating Body Surface Area (BSA) and at 50mg/m²BSA is infused intravenously (2mg/ml).

BSA (M2) = SQ. ROOT OF [(HEIGHT (CM) x WEIGHT (KG) / 3600)]

Collection of blood and pleural liquid:

The Pleural liquid of the patient was collected by the nursing professional and immediately introduced into collection bottles. Approximately 100ml aliquot from each patient was collected and immediately used for all the biomarkers and stored at -20°C. The blood of the patient was collected by the nursing professional and immediately introduced into collection tubes. The serum was collected after centrifugation at 4000rpm at room temperature and immediately used for all the biomarkers and stored at -20°C.

Estimation of the biomarkers:

Level of pleural markers and serum markers were estimated by using ELISA kits according to manufacturer’s instructions. The pleural thickness was identified by measuring the optical density in a spectrophotometer.

Quantification of MDA, ROS generation and hydroperoxide levels:

The extent of lipid peroxidation was measured in terms of MDA¹⁰. by incubating an aliquot with 1ml of TBA reagent (0.3% Thiobarbituric acid, 0.3% Sodium Dodecyl Sulphate and 7.5% acetic acid pH 3.5) in boiling water for 20 minutes. At 532nm, the optical density was measured, quantified against tetra methoxy propane (TMP, equimolar to MDA) standard curve. The results are expressed as nmol MDA formed/mg protein.

Generation of Reactive oxygen species (ROS) was determined (Eruslanov and Kusmartsev 2010) by following DCF fluorescence. Briefly, an aliquot (0.1mg protein) was incubated for 30 minutes at room temperature in Locke's solution (pH 7.4; in mM: NaCl-154, KCl-5.6, NaHCO₃-3.6, HEPES-5, CaCl₂-2, and glucose-10) containing 5M DCFDA (excitation: 480 nm; emission: 530nm). The amount of ROS produced was calculated using a DCF standard curve and expressed as pmol DCF/min/mg protein.

Based on a recycled ferrous ion-mediated oxidation of xylenol orange, the amounts of hydroperoxides (HP) were determined¹¹. Test sample (20µg protein) In a 96-well microtiter plate, 200l of FOX1 reagent (in M: Xylenol orange-100, ammonium ferrous sulphate-250, sorbitol-100, 25mM H₂SO₄) was allowed to react for 30 minutes at room temperature with 200l of FOX1 reagent (in M: Xylenol orange-100, ammonium ferrous sulphate-250, sorb At 560nm (€ -1.5X104mol/cm), the color generated was measured and expressed as nmol hydroperoxides (HP)/mg protein.

Estimation of activity levels of antioxidant enzymes:

The activity of superoxide dismutase (SOD) was assessed using an indirect technique that monitors Quercetin autooxidation inhibition¹². Quercetin (100M) is introduced to a reaction mixture of phosphate buffer (0.016M, pH 7.8, including TEMED-4mM and EDTA 0.04mM) and its rate of oxidation is monitored at 406nm for 3 minutes. One unit of the enzyme and activity represented as units/mg protein is defined as the ability of the test sample to inhibit quercetin oxidation by half.

Catalase activity was measured¹³. by following the hydrolysis of hydrogen peroxide at pH 7.2 at 240nm using a spectrophotometer.

The activity of glutathione-S-Transferase (GST) was measured at 340nm by following glutathione conjugation to CDNB¹⁴. The reaction was started by adding an aliquot (0.01mg protein) of the test sample to phosphate buffer (0.1M, pH 6.5 containing in mM: EDTA-0.5, CDNB-0.075, GSH-0.05). The activity was measured as nmol conjugate formed/min/mg protein (€ -9.6/mM/cm) by measuring the increase in optical density at 340nm over 3 minutes.

Statistical analysis:

The data were analyzed using GraphPad Prism 5.0 software and a one-way ANOVA followed by a post hoc Tukey's test to compare the control and treatment groups as well as among the groups (p 0.05). Different alphabet letters indicate significance difference among the respective groups. In some assays, *indicates significance difference from control (p≤0.05).

RESULTS AND DISCUSSION:

Numerous chemotherapy studies have focused on limited parameters like tumor suppression and likeliness of cancer relapse. However, the studies addressing the oxidative status of the patients are limited. Additionally, the exclusive reports about the biochemical indices of the pleural liquids are near to none. Hence, we set out to study the changes in the redox markers in the serum and pleura of the ovarian cancer patients during the chemotherapy treatments.

The demographic data about the patients volunteered are listed in table 1. Nearly half of them had key symptoms like nausea and pain. Most patients with ovarian cancer are reported to complain of abdominal pain as reported earlier.¹⁵

MATERIALS AND METHODS:

Participants and enrollment:

Drug and Dosage:

BSA (M2) =

Sq. root of [(Height (cm) x Weight (kg) / 3600)]

Collection of blood and pleural liquid:

Estimation of the biomarkers:

Quantification of MDA, ROS generation and hydroperoxide levels:

Generation of Reactive oxygen species (ROS) was determined (Eruslanov and Kusmartsev 2010) by following DCF fluorescence. Briefly, an aliquot (0.1mg protein) was incubated for 30 minutes at room temperature in Locke's solution (pH 7.4; in mM: NaCl-154, KCl-5.6, NaHCO3-3.6, HEPES-5, CaCl2-2, and glucose-10) containing 5M DCFDA (excitation: 480 nm; emission: 530nm). The amount of ROS produced was calculated using a DCF standard curve and expressed as pmol DCF/ min/ mg protein.

Based on a recycled ferrous ion-mediated oxidation of xylenol orange, the amounts of hydroperoxides (HP) were determined¹¹. Test sample (20µg protein) In a 96-well microtiter plate, 200l of FOX1 reagent (in M: Xylenol orange-100, ammonium ferrous sulphate-250, sorbitol-100, 25mM H2SO4) was allowed to react for 30 minutes at room temperature with 200l of FOX1 reagent (in M: Xylenol orange-100, ammonium ferrous sulphate-250, sorb At 560nm (€ -1.5X104mol/cm), the color generated was measured and expressed as nmol hydroperoxides (HP)/ mg protein.

Estimation of activity levels of antioxidant enzymes:

Catalase activity was measured¹³. by following the hydrolysis of hydrogen peroxide at pH 7.2 at 240nm using a spectrophotometer.

The activity of glutathione-S-Transferase (GST) was measured at 340nm by following glutathione conjugation to CDNB¹⁴. The reaction was started by adding an aliquot (0.01mg protein) of the test sample to phosphate buffer (0.1M, pH 6.5 containing in mM: EDTA-0.5, CDNB-0.075, GSH-0.05). The activity was measured as nmol conjugate formed/ min/ mg protein (€ -9.6/mM/cm) by measuring the increase in optical density at 340nm over 3 minutes.

Statistical analysis:

RESULTS AND DISCUSSION:

Table 1. Demographic parameters of the participants who enrolled themselves for the biochemical studies.

S. No.	Parameter (n=40)	Range/ Percent
1	Age (years)	43-59 years
2	Body weight (kg)	41.5-91.4 kg
3	Height (cm)	148-162 cm
4	Pain	27 patients out of 40
5	Nausea	25 patients our of 40

Severely affected energy metabolism is an established pathophysiology in the cancer patients. The glucose and cholesterol levels are highly altered among the patient serum. Similarly, in our study, the patient sera demonstrated extremely higher levels of glucose and lower levels of cholesterol (Fig 1). According to research, glucose levels in cancer patients could be a useful prognostic indication. Increased expression of GLUT1, a trans-membrane protein involved in glucose uptake in ovarian tumors, is linked to a shorter survival time in ovarian cancer patients.¹⁶. There was no change in the level of fasting glucose in the serum samples before and after the chemotherapy session. The random blood sugar was marginally reduced after chemotherapy sampling. There was a very huge increase (38%) in the levels of total cholesterol among serum samples with the chemotherapeutic analyses among ovarian cancer patients. These effects are observed in the serum levels of total cholesterol, however, very few studies have reported this effect. Interestingly in lines with the serum results, the total cholesterol among pleural samples was greatly increased. The random and fasting sugar levels among the pleural liquids were significantly reduced. However, this is the first report about the glucose levels in the pleural liquids among the ovarian cancer patients. Interestingly the levels were not changed after the chemotherapy cycle. We believe, these physiological markers cannot be altered with a single dose of chemotherapy drug. It is proposed that multiple chemotherapy cycles as required by the patient may bring the levels down to normalcy.

The levels of TNF alpha increased (57%) after chemotherapy among serum samples of ovarian cancer patients. The enzymic levels of Lactic Dehydrogenase were significantly decreased (53%) among post chemotherapy serum samples. The oldest marker for cancer physiology, LDH were reduced with treatment among pleural samples. The pleural liquid density was not altered among the treatment groups (Figure 2). The reduction in the LDH levels show the protective effects of chemotherapy¹⁷. Our study also indicates the improved TNF alpha levels which monitor the carcinogenesis. In addition, the increased GST levels show the responsiveness of the liver in relation to the foreign body that is drug.

Oxidative stress has embroiled in the causes of several diseases, including cancer. Changes of the cellular redox balance modify the initiation, upgrading, and progression of tumor cells¹⁸^,¹⁹. The continued synthesis of oxidants and free radicals has an impact on cellular systems that regulate cell proliferation and apoptosis, which plays a key role in the beginning and progression of cancer. Oxidants can originate and promote the oncogenic phenotype or trigger apoptosis, depending on the levels of ROS and RNS in the cellular environment, and hence act as antitumor agents.

The antioxidant enzyme activity was markedly reduced (81%) after chemotherapy among ovarian cancer patients (Figure 3). The SOD activity levels significantly increased with the treatment and up to 28% among ovarian cancer patients. Interestingly there was a remarkable raise in the levels of ROS among the serum of patients after the treatment. However, the increase in the SOD could not be explained with these data. The level of oxidative marker hydroperoxides estimated by FOX1 reagent was shown to decrease after treatment among patients. Similar with the serum, the lipid peroxides level among pleural samples were reduced significantly among treatment groups. Mechanisms involving oxidants and antioxidant enzymes and compounds maintain homeostasis between the generation and elimination of oxidants.

If this balance is changed, it will lead to an improved state of oxidative stress that change the key biomolecules and cells of living organism. Serum malondialdehyde (MDA) was measured as an indicator of lipid peroxidation and antioxidant status was assessed by estimating serum vitamin E and erythrocyte superoxide dismutase (SOD) levels. Finally, the status of antioxidants and lipid peroxidation was correlated with the pathophysiology of ovarian cancer. The antioxidant enzyme catalase was reduced among pleural liquid samples with treatment.

Figure 1. Effect of chemotherapy on the glucose levels in serum and pleural liquids. Different letters indicate significant difference at P≤ 0.05.

Figure 2. Effect of chemotherapy on the levels of non-specific cancer markers in serum and pleura. Different letters indicate significant difference at P≤ 0.05.

Oxidative markers hydroperoxides and lipid peroxides were markedly reduced with treatment among pleural samples. The ROS levels were surprisingly increased among pleural samples with treatment (Table 2).

Table 2. Modulatory effects of chemotherapy on the pleural oxidative markers. Different letters indicate significant difference at P≤ 0.05.

	Before chemo ± SE	After Chemo ± SE
Catalase	45.5 ± 2.05 a	12.1 ± 0.55 b
ROS	26.3 ± 1.19 a	128.3 ± 5.79 b
SOD	78.8 ± 3.56 a	131.3 ± 5.93 b
HP	56.6 ± 2.55 a	33.3 ± 1.50 b
LPO	15.2 ± 0.68 a	9.1 ± 0.41 b

Figure 3. Effects of chemotherapy on the oxidative markers in the serum. Different letters indicate significant difference at P≤ 0.05.

Specific antigen levels have been strongly indicated with the severity of various cancers²⁰. Additionally, the major cancer marker Carcinoembryonic antigen was greatly reduced with treatment among pleural samples. There is a newfound clinical significance for CA125 in terms of better sensitivity and specificity in identifying epithelial ovarian cancer relapse. Nearly three decades following the discovery of CA125, it is still highly recommended to monitor the response to the chemotherapy among the ovarian cancer patients as well as to detect the relapse²¹. The major cancer marker Cancer antigen 125 was greatly reduced with treatment among pleural samples (Figure 4). The additional cancer marker Cancer antigen 27.25 was marginally reduced with treatment among pleural samples. As anticipated the levels of Carcinoembryonic antigen levels were greatly reduced among post treatment samples. The treatment of ovarian patients with the drug resulted in the fall of the major marker Cancer antigen 125 markedly among serum samples. Interestingly there was a marginal but significant reduction among CA27.29 antigen among post treatment samples. Reduction in the specific cancer markers among serum and pleural samples among chemotherapy group clearly indicate the positive effects of chemotherapy in these patients.

Figure 4. Effect of chemotherapy on the specific cancer markers among serum and pleura. Different letters indicate significant difference at P≤ 0.05.

The presence of peritoneal metastases is always limited to recurring or developing disease, although pleural metastases have been known to be discovered at the time of initial diagnosis.

Figure 5. Changes in the pleural proteins and thickness among chemotherapy patients. Different letters indicate significant difference at P≤ 0.05.

Furthermore, the recent discovery of ovarian cancer stem cells in ascites, which exhibit characteristics similar to conventional cancer stem cells, is a new contributing factor to not just metastasis but also chemo-resistance. There was a significant reduction in the total protein content of the pleural liquid specimens after treatment. The pleural liquid density was not altered among the treatment groups (Figure 5).

CONCLUSION:

The pleural and serological analyses before and after platinum chemotherapy session demonstrated wide range of alterations in biochemical and protein markers among ovarian cancer patients. However, the pleural thickness and the pleural levels of the cancer biomarkers were not impressively reduced post chemotherapy. According to previous research, a combination of antioxidants and chemotherapy has a considerable effect on the sensitivity of cells to chemotherapy. The detection of chemo-resistance targets through biomarkers and/or screening potency will have a substantial impact on the treatment of this disease. Hence, our data can be considered as a pavement for the discovery of further markers in the pleural liquid and serum as well as a reliable set of tests for monitoring chemotherapeutic response.

CONFLICT OF INTEREST:

Author declares that there is no conflict of interests.

REFERENCES:

1. Klymenko, Y. and Nephew, K. P. Epigenetic crosstalk between the tumor microenvironment and ovarian cancer cells: a therapeutic road less traveled. Cancers (Basel). 10, 295 (2018). https://doi.org/10.3390/cancers10090295

2. Marí-Alexandre, J. et al. Interplay between microRNAs and oxidative stress in ovarian conditions with a focus on ovarian cancer and endometriosis. Int. J. Mol. Sci. 20, 5322 (2019). https://doi.org/10.3390/ijms20215322

3. Nikolova, N. K. Introduction to microwave imaging. (Cambridge University Press, 2017). doi:10.2528/PIERB17081003

4. Winarno, N., Rusdiana, D., SUSİLOWATİ, E. & AFİFAH, R. M. A. Implementation of integrated science curriculum: a critical review of the literature. J. Educ. Gift. Young Sci. 8, 795–817 (2020). https://doi.org/10.17478/jegys.675722

5. Worzfeld, T. et al. Proteotranscriptomics reveal signaling networks in the ovarian cancer microenvironment. Mol. Cell. Proteomics 17, 270–289 (2018). DOI:https://doi.org/10.1074/mcp.RA117.000400

6. Singh, J. et al. Genetic diversity and drug susceptibility profile of Mycobacterium tuberculosis isolated from different regions of India. J. Infect. 71, 207–219 (2015). https://doi.org/10.1016/j.jinf.2015.04.028

7. Principe, N. et al. Malignant Pleural Effusions—A Window Into Local Anti-Tumor T Cell Immunity? Front. Oncol. 11, 1422 (2021). https://doi.org/10.3389/fonc.2021.672747

8. Flieger, J., Flieger, W., Baj, J. & Maciejewski, R. Antioxidants: Classification, natural sources, activity/capacity measurements, and usefulness for the synthesis of nanoparticles. Materials (Basel). 14, 4135 (2021). https://doi.org/10.3390/ma14154135

9. Bryant, A. et al. Impact of residual disease as a prognostic factor for survival in women with advanced epithelial ovarian cancer after primary surgery. Cochrane Database Syst. Rev. (2021). https://doi.org/10.1002/14651858.CD015048

10. Ohkawa, H., Ohishi, N. & Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358 (1979). https://doi.org/10.1016/0003-2697(79)90738-3

11. Wolff, L. Inventing Eastern Europe: The map of civilization on the mind of the Enlightenment. (Stanford University Press, 1994). https://doi.org/10.1515/9780804765299

12. Kostyuk, V. A. & Potapovich, A. I. Superoxide-driven oxidation of quercetin and a simple sensitive assay for determination of superoxide dismutase. Biochem Int 19, 1117–1124 (1989). DOI:https://doi.org/10.1128/MCB.19.1.2

13. Aebi, H. [13] Catalase in vitro. in Methods in enzymology vol. 105 121–126 (Elsevier, 1984). https://doi.org/10.1016/S0076-6879(84)05016-3

14. Mannervik, B. et al. Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties. Proc. Natl. Acad. Sci. 82, 7202–7206 (1985). https://doi.org/10.1073/pnas.82.21.7202

15. Woopen, H. et al. Prognostic role of chemotherapy-induced nausea and vomiting in recurrent ovarian cancer patients: results of an individual participant data meta-analysis in 1213. Support. Care Cancer 28, 73–78 (2020). DOI: https://doi.org/10.1007/s00520-019-04778-1

16. Liu, H., Simonyan, K. and Yang, Y. Darts: Differentiable architecture search. arXiv Prepr. arXiv1806.09055 (2018). https://doi.org/10.48550/arXiv.1806.09055

17. Torrey, H. et al. Targeting TNFR2 with antagonistic antibodies inhibits proliferation of ovarian cancer cells and tumor-associated Tregs. Sci. Signal. 10, eaaf8608 (2017). DOI: 10.1126/scisignal.aaf8608

18. Scutiero, G. et al. Oxidative stress and endometriosis: a systematic review of the literature. Oxid. Med. Cell. Longev. 2017, (2017). https://doi.org/10.1155/2017/7265238

19. Hou, J., Inganäs, O., Friend, R. H. and Gao, F. Organic solar cells based on non-fullerene acceptors. Nat. Mater. 17, 119–128 (2018). https://doi.org/10.1038/nmat5063

20. Phelan, C. M. et al. Identification of 12 new susceptibility loci for different histotypes of epithelial ovarian cancer. Nat. Genet. 49, 680–691 (2017). https://doi.org/10.1038/ng.3826

21. Bottoni, P. and Scatena, R. The role of CA 125 as tumor marker: biochemical and clinical aspects. Adv. Cancer biomarkers 229–244 (2015). https://doi.org/10.1007/978-94-017-7215-0_14

Received on 20.03.2022 Modified on 30.04.2022

Research J. Pharm. and Tech 2022; 15(12):5665-5671.

DOI: 10.52711/0974-360X.2022.00955