Inositol hexakisphosphate induces apoptosis, cell cycle arrest in non-Hodgkin’s Burkitt lymphoma cells and mediates anti-angiogenic, antitumor effects in T-cell lymphoma bearing Swiss albino mice

2.1 Cell culture

Burkitt’s lymphoma cells Raji were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 50 IU/mL penicillin, and 50 mg/mL streptomycin in a humidified atmosphere of 5% CO₂ at 37 °C.

2.2 Detection of apoptotic cell death by flow cytometry

All experiments were performed using cells after 24 hrs when the confluence of 2 × 10⁶ cells/mL was reached in six-well plates. Later, varying concentrations of IP6 (1 μM, 3 μM and 5 μM) were loaded onto each well for 24, 48 and 72 hrs. After treatment, the medium was removed and the cells were washed with 1X PBS and trypsinated for 3–5 mins at 37 °C. Following this, the cells were centrifuged at 2000 rpm for 5 mins at 4 ℃, washed with 1X PBS and re-suspended in 100 μL/well of the 1X binding buffer. Annexin V-FITC (1 μL/well) and 1 μL of propidium iodide were added to stain the cells after which the fluorescence was analysed by flow cytometry (A00-1–1102, Beckman Coulter, USA).

2.3 Cell cycle analysis

After a confluence of 3 × 10⁵ cells/mL was reached, varying concentrations of IP6 were added at varying time durations as mentioned earlier. The cells were washed with 1X PBS, trypsinated, centrifuged at 2000 rpm at 4 ℃ for 5 mins. After centrifugation, the pellet was re-suspended in 500 μL of 70% ice-cold ethanol and incubated for 2 hrs at 4 ℃. The cells were washed with PBS, re-suspended in 100 μL propidium iodide/RNaseA solution (cell cycle buffer). After an incubation of 30–60 mins, the cells were analysed by flow cytometry (A00-1–1102, Beckman Coulter, USA).

2.4 Institutional approval, animal maintenance and in vivo experiment

The animal study was approved by Institutional Animal Ethics Committee of Bharathidasan University and adhered to the CPCSEA regulations. Adult male Swiss albino mice (30–35 gm) were housed under controlled temperature and hygiene under sanitized conditions in polypropylene cages with sterile paddy husk in normal day and night (12-hour light: 12-hour dark) conditions. Standard laboratory diet (22% crude protein, 4.12% crude oil, 2.79% crude fiber, 7.81% ash and 1.34% sand silica) and water were used to feed the animals.

Eighteen mice were randomized into 3 groups of six animals each: normal, DAL induced and DAL induced mice treated with IP6 at a dose of 300 mg/kg. The animals injected with DAL cells, were kept as such for three days. Three days into the experimental period, the animals were administered with IP6 every day, intraperitoneally for a period of ten days. Thus, the total experimental period was 13 days. Body weight was measured and recorded from all animals every day from the start to the end of the experiment. The antitumor effects of IP6 were assessed after 10 days of treatment, following euthanasia, at the point considered to be the end of the experiment. Intraperitoneal ascites fluid was collected from the induced and treated groups at the end of the trial and the tumor volume was ascertained in tumor-induced and treated animals. The degree of blood vessel growth and formation was used to determine angiogenesis.

2.5 Trypan blue exclusion method for assessment of cytotoxicity in vitro

Sterile PBS was injected and DAL cells were collected into a Petri dish from the peritoneal region of tumor-bearing mice. The cells were incubated at 37 °C for 2 h, followed by aspiration performed to dislodge the non-adherent cells. The cell suspension was then treated with IP6 (0.5 and 1 mg/mL, which resembles 150 and 300 mg/kg BW in mice). Later, cell viability was determined using trypan blue staining and the treated cells were observed using a hemocytometer (Shrivastava and Ganesh, 2010, Sriram et al., 2010).

2.6 Analysis of in vivo parameters

Blood collected from the animals of each group was used to separate the serum by centrifuging at 2500 rpm for 15 min. The separated serum was used for biochemical, hematological and antioxidant assays. Assay of biochemical parameters include the analysis of serum glutamate oxalate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP) levels. RBC, WBC and hemoglobin were the hematological parameters analysed. Serum levels of cellular metabolite lactate dehydrogenase (LDH), enzymatic antioxidant superoxide dismutase (SOD) and non-enzymatic antioxidant glutathione (GSH) were measured. Lipid peroxidation marker malondialdehyde (MDA) was measured using centrifugation of the supernatant collected from the liver homogenate at 12000 rpm for 30 min (Antony et al., 2013, Zhang et al., 2019).

2.7 Histopathology

Liver tissue was fixed in 10% formalin for further use. Paraffin sections of 5 µm thickness were prepared, stained with hematoxylin and eosin, observed under a microscope and photographed at 40X magnification (Ye et al., 2009).

2.8 Statistical Analysis

All data were expressed as mean ± SD. The statistical significance was assessed by one- way analysis of variance (ANOVA) using SPSS version 17 and the individual comparisons were obtained by Duncan’s multiple range test (DMRT). A value of p < 0.05 was considered to be statistically significant among the groups. Alike alphabets in lower-case indicate no significant change, however, unlike alphabets specify significant change between the groups (Zar, 1984).

3.1 IP6-induced apoptotic cell death in Raji Cells

Apoptosis is a normal mechanism for maintaining homeostasis in cells during development and aging. There are several stimuli that can lead to an apoptotic trigger. Although all these stimuli cannot lead to cell death, interaction with anticancer drugs can result in cell death through apoptosis in a p53 dependent mechanism (Elmore, 2007). The Annexin V-FITC/PI staining is an appropriate and subtle method to detect cellular apoptosis by detecting the stage of apoptosis (early or late) by the virtue of plasma or nuclear membranes (Rieger et al., 2011, Filograna et al., 2015).

In this study, the early apoptotic cells increased from 2.42% in control to 4.03% in cells treated with 1 µM IP6. This percentage increased considerably to 4.52% and 5.01% when treated with 3 µM and 5 µM IP6 after 24 hrs. The late apoptotic cells increased from 0.69% in control to 0.99% (1 µM), 1.03% (3 µM) and 1.11% (5 µM) in treated cells. After treatment of 72 hrs, the early apoptotic cells increased from 1.78% in control to 2.31% (1 µM), 2.88% (3 µM) and 3.03% (5 µM) in treated cells. The late apoptotic cells increased from 0.77% in control to 1.45% in 5 µM IP6 treated cells [Fig. 1]. These results indicate that apoptosis was effectively induced in Raji cells treated with IP6.

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Fig. 1

IP6 induces apoptosis in Raji cells as evidenced by Annexin V-FITC/PI staining using flow cytometry. P value of<0.05 (*P < 0.05, **P < 0.01, compared with the control group) are considered significant.

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3.2 IP6-induced cell cycle arrest at G2/M phase

Cell cycle arrest and apoptosis occur as a result of the activation of p53 which may prevent tumor cell from developing (Chen, 2016). The cell cycle plays key roles in replication of a cell, cell death and its functioning (Ramadoss and Sivalingam, 2021). In the first phase called growth 1 phase or gap 1 phase (G1), the cells will produce new proteins and enlarge in size until it reaches a G1 checkpoint (Maira et al., 2007). The S phase of synthesis phase occurs between the G1 phase and G2 phase (Growth 2 or Gap 2 phase) where the DNA replication occurs for multiplying the genetic content (Fischer et al., 2018). The cells at G2 phase grow rapidly, synthesize proteins and prepares itself for mitotic phase (Li et al., 2015). Connecting to this, several cell types have modified the expressions of regulatory molecules such as cyclins and cyclin-dependent serine/threonine kinases (CDKs) to regulate the cell cycle. Novel anticancer agents are targeted towards these molecules for better management of diseases such as cancer. In this view, intervening or causing cell cycle arrest may be a promising approach for cancer therapy (Karthi et al., 2016, Guo et al., 2015). Analysis of cell cycle by flow cytometry can determine the phase of cell cycle in which the cells being studied are part of (Pozarowski and Darzynkiewicz, 2004).

Although a large population of cells were part of the G0/G1 phase, and the increase in percentage was time- and dose-dependent, the changes were not significant in comparison to the control. After 24 hrs of treatment with IP6, significant increase in occurrence of cells at G2/M phase was observed at doses of 3 µM and 5 µM in comparison to the control [Fig. 2A]. The percentage of cells at G2/M and S phases decreased with increasing doses of IP6 compared to the cells at G0/G1 phase. Yet, the increase in percentage of cells at G2/M phase was significant when compared to the percentage of control cells after 72 hrs [Fig. 2C]. This indicates that IP6 could induce cell cycle arrest at G2/M phase significantly in Raji cells after 72 hrs at all three doses tested.

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Fig. 2

IP6 induced cell cycle arrest at G2/M phase in Raji cells as analysed by flow cytometry. P value of<0.05 (*P < 0.05, **P < 0.01, compared with the control group) are considered significant.

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Drugs or compounds which can decrease the viability of neoplastic cells, induce cell cycle arrest and apoptosis in such cells are considered effective anticancer agents (Lee et al., 2020). Detection of apoptosis by Annexin V-FITC/PI assay and the cell cycle arrest at G2/M phase indicated that IP6 is an effective anti-neoplastic entity.

3.3 In vitro analysis of cell death by trypan blue staining

Thousands of ethnomedical plants are explored every year which are known to possess potent anticancer effects (Dai and Mumper, 2010). Cytotoxic agents isolated from these plants can both inhibit cell proliferation and result in cell lysis thereby forming the basis for treatment of malignancies (Sui et al., 2013, Seca and Pinto, 2018). Trypan blue staining is an exclusion technique in which dead cells are stained blue, whereas, the viable cells are not, because of their intact membranes. This happens as a consequence of apoptosis and loss of membrane integrity among dead cells (Khairunnisa and Karthik, 2014). According to the trypan blue staining, increased cell death was observed among DAL cells treated with 300 mg/kg of IP6. Therefore, 300 mg/kg of IP6 was used for in vivo experimentation [Fig. 3].

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Fig. 3

Cell viability assessment by trypan blue staining using various doses of IP6 at 40X magnification.

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3.4 Tumor burden altered significantly after IP6 treatment

Changes were observed in body weights of treated and control animals. Body weights of IP6 treated mice decreased significantly in comparison to the DAL induced mice [Fig. 4A]. There was a reduction in tumor volume [Fig. 4B]. This reduction in body weight was linked to decrease in number of viable tumor cells and the volume of ascitic fluid, which is a necessity to achieve nutritional sustainability among tumor models. This is an indication of systemic or local cytotoxic effects (Dolai et al., 2012, Samudrala et al., 2015). A drop in tumor volume is a critical parameter for identification of an effective anticancer drug (Kathiriya et al., 2010).

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Fig. 4

Tumor burden of the experimental animals before and after treatment with IP6 (a) changes in body weight (b) changes in tumor volume.

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3.5 IP6 curbed angiogenesis in tumor-induced mice

The formation of new blood and lymphatic vessels, necessary for tumor metastasis, occur as a result of angiogenesis and lymphangiogenesis. This system supplies nutrients, oxygen, helps in removal of waste products and supports tumor cells to metastasize. When the vascular system does not give the cancer cells the provision of such supply, they die due to necrosis or apoptosis. Angiogenesis occurs in several stages among tumor cells that include membrane destruction and resultant hypoxia as the preliminary step. The next steps involve the migration, proliferation (once every 1000 days) and stabilization of endothelial cells that are activated by the influence of angiogenesis (Nishida et al., 2006, Yoo and Kwon, 2013, Rajabi and Mousa, 2017). Controlling or inhibiting angiogenesis is a key approach in cancer treatment (Lee et al., 2015). The identification of drugs or compounds with anti-angiogenic activity can help reduce the adverse effects of drugs intended to be used as antitumor medications (Abdallah et al., 2018). Post-dissection, the peritoneal region was observed for vessel growth, which indicated enlarged or increased growth of capillaries in DAL mice. The treatment with IP6 restricted the vessel growth in tumor induced mice. This indicates the anti-angiogenic effect of IP6 [Fig. 5].

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Fig. 5

Anti-angiogenic effects as observed by changes in vessel growth of the peritoneal regions of normal, DAL-induced and IP6 treated mice.

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3.6 Hematological changes were significant after IP6 treatment

Myelosuppression and anaemia, arise mutually as a result of reduction in RBC and percent hemoglobin due to deficiency of iron or other hemolytic conditions. These are major parameters to be addressed during cancer therapy and are prognostic factors for progression of lymphoma (Ghosh et al., 2011, Hasenclever et al., 1998, Knight et al., 2004). Leukocytosis is a determining factor for inflammation and is representative for oncogenesis and progression (Iida et al., 2012, Kleppe et al., 2018). Declined RBC and hemoglobin levels along with elevated WBC levels were observed in this study. This indicates that there was a significant therapeutic effect of IP6 on myelosuppression, anaemia and leukocytosis in DAL mice by creating a profile of typical hematology [Fig. 6].

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Fig. 6

Changes in hematological parameters of the experimental animals before and after treatment with IP6 (a) RBC (b) WBC (c) Hemoglobin.

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3.7 Biochemical parameters altered significantly after IP6 treatment

Although liver is far from tumor-affected peritoneum, it is affected by disorders such as cancer. Liver seems to be the primary site preferred for such metastatic changes (Seyfried and Huysentruyt, 2013). High levels of hepatic transaminases such as ALP, SGOT and SGPT are indicative of liver damage in such cases (Oh et al., 2020b, Zachariah et al., 2017, Haldar et al., 2010). The high levels of these transaminases in DAL mice reverted to near-normal in IP6 treated group [Table 1A].

Cancer cells can generate excessive levels of hydrogen peroxide and the resultant reactive oxygen species (ROS) which can cause mutations and cause injury to normal cells leading to oncogenesis (He et al., 2017, Han and Chen, 2013). Dietary antioxidants can convert these free radicals into wastes or by-products that are eventually released by the body (Esquivel-Chirino et al., 2013). SOD, is an enzymatic antioxidant which can catalyse the simultaneous oxidation and reduction of superoxide anion to hydrogen peroxide. This peroxide is later detoxified into oxygen and water by enzymes and other components of antioxidant system (Asakura and Kitahora, 2018). GSH, a non-enzymatic antioxidant can play dual roles in cancer. On the positive note, it can scavenge free radicals and aid as an adjuvant for enzymes involved in detoxification of carcinogens (Balendiran et al., 2004, Sznarkowska et al., 2017). Therefore, a failure in antioxidant defence can lead to redox imbalance that cancer cells relish on (Snezhkina et al., 2019). The failure in antioxidant defence observed among DAL mice was reformed to normalcy in IP6 treated group [Table 1B and 1C].

MDA is an end-product of oxidative degeneration which can result in the release of toxic substances such as hydroperoxides through lipid peroxidation (Helbock et al., 1993). Treatment with IP6 reduced the MDA levels significantly which signifies the reduction in lipid peroxidation of mice induced with lymphoma [Table 1D]. LDH is a standard diagnostic marker for violent forms of cancers such as lymphoma, which indicates the intracellular metabolism, mode of glycolysis and neoplastic conversion. It is generally augmented in cancer patients and protects tumor cells from hypoxia‐induced necrosis [Table 1E] (Forkasiewicz et al., 2020, Jurisic et al., 2015, Feng et al., 2018). Reduction in lipid peroxidation and an increase in antioxidant system that possess SOD and GSH shows the potential of IP6 in inhibition of DAL-induced intracellular oxidative stress that occurred as a result of damage induced by free radicals (Thavamani et al., 2014).

3.8 Significant histological changes after IP6 treatment

The cells of livers of normal mice showed well-defined cytoplasm, prominent nucleus and distinct central vein. Centrilobular hepatic necrosis, vacuolization, disintegrated cell membranes and disintegrated central vein with dilated sinusoids were the damages observed in the livers of DAL induced mice. The damages observed in architecture of livers of DAL mice were revived to normalcy in mice treated with IP6 [Fig. 7]. This elucidates the protective effects of IP6 on liver after tumor induction.

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Fig. 7

Histopathological examinations of livers of normal, DAL-induced and IP6 treated mice at 40X magnification.

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