INTRODUCTION:

Cancer causes abnormal cells to develop and multiply uncontrollably¹. About 13% of global deaths are cancer-related². The second biggest cause of cancer-related deaths in the world is colorectal cancer (CRC). Globally, it was estimated that there were around 1.9 million new instances of colorectal cancer and more than 0.93 million mortalities from the disease in 2020³. For males and women, respectively, the lifetime risk of colorectal cancer is around 1 in 21 (4.7%) and 1 in 23 (4.4%)⁴.

This is the third most prevalent cancer diagnosed in the USA, after skin malignancies. According to the American Cancer Society projected thatin 2023, there may be around 1.06 million new cases of CRC. The global new CRC cases is predicted to reach 3.2 million in 2040⁵.

Over the past 20 years, significant improvements in colorectal cancer therapy have been made, enhancing early diagnosis and multimodal care, as well as our understanding of the biology of the illness and the processes behind tumor growth⁶. As a result, compared to prior times, colorectal cancer incidence and fatality rates have been low⁷. In cancer progression, various inflammatory cytokines (IL-6, IL-1β, TNF-a) and pathways (JAK/STAT) are involved⁸. Nowadays researchers are mainly focusing on reducing the expression of these inflammatory cytokines andpathways involved in cancer progression and metastasis.

Gefitinib (GEF), a popular anticancer drug that selectively inhibits EGFR tyrosine kinase⁹, has been used to treat lung cancer on a large scale, either alone or in conjunction with other drugs¹⁰. Gefitinib has considerably decreased colony formation, migration, and cell proliferation in vitro in colorectal cancer¹¹. Clinical studies using GEF as a single or combined treatment for colorectal malignancies demonstrated encouraging outcomes¹². Even though GEF is effective in treating colon cancer, it also has certain dose-related adverse effects like all other cytotoxic medications¹³. Therefore, any effort to lower the GEF dose would result in a significant decrease in the comorbidities related to its usage. The NLC loaded with GEF may have excellent sensitivity in cancer cells with low doses, according to our research's hypothesis. NLCs (Nanostructured lipid carriers) are made up of solid nanoparticles in aqueous dispersions that are stabilized by one or two surfactants and comprise a combination of liquid and solid lipids¹⁴. Nanotechnology developments are crucial to the treatment of cancer and ultimately raise the survival rate of cancer patients. Nanotechnology improves the results of traditional methods and plays a significant role in cancer diagnosis, integrative therapy, and diagnosis. As an effective anti-cancer drug, the structural morphology, shape, and size of nanocarriers are crucial in cancer therapy^15,16. Effective anti-viral, anti-cancer, anti-bacterial, and even anti-inflammatory drugs may be delivered in the form of nanocarriers^17,18. Numerous nanomaterials have already been studied and have proven advantageous for cancer research^19,20.

Therefore, the goal of this work was to perform in vitro testing to evaluate how effective it is at causing cytotoxicity. The effect of Nano-GEF was investigated on cell death and apoptosis using morphology examination, MTT assay, qualitative and quantitative DNA fragmentation assay, and gene expression of interleukin cytokines IL-6, IL-6R, gp130, Bcl-2,Bax, NF-kB,and JAK-STAT by qRT-PCR.

MATERIALS AND METHOD:

Gefitinib was purchased from LC Laboratories (Woburn, MA, USA). Tween-80 (T80) was purchased from LobaChemei (Mumbai, India). Sodium lauryl sulfate (SLS) and stearic acid (SA) were purchased from Himedia (Mumbai, India). Sesame oil was obtained from a local merchant (Jazan, Saudi Arabia). Primers were purchased from Macrogen (Seoul), Kits for RNA extraction (Bio-Rad), cDNA reverse transcription kits, and SYBER green was purchased from Applied Biosystem (USA).

Preparation of Nano-GEF:

The previous project was an inspiration for this one²¹. It was prepared by mixing two phases namely lipid and aqueous phase. To create the lipid phase for the GEF-loaded NLC (Nano-GEF), stearic acid and sesame oil were used. To make the aqueous phase, Tween-80 and sodium lauryl sulfate were dissolved in water. An amount of stearic acid (500 mg) was placed in a beaker and melted on a hot plate. Sesame oil (250 L) was then added to the melted stearic acid and well-mixed. Tween-80 (75 µL) and sodium lauryl sulfate (25 mg) were mixed with 25 mL of Millipore water in a separate beaker. Water was heated to a temperature of 70 degrees Celsius. A homogenizer was used to mix the aqueous and lipid phases at a speed of 5000 rpm for 20 minutes. Whatman cellulose filter paper (Macherey-Nagel, GmbH, Duren, Germany) was used to filter it, and the filtered liquid was collected in a clean container for further testing.

Cell culture:

King Abdulaziz University, Jeddah, Saudi Arabia, graciously gave us the HCT116 cell lines that they had purchased from ATCC. These cell lines were grown in full RPMI 1640 medium with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine added as supplements. All cells were incubated at 37°C in a humid atmosphere of 95% air and 5% CO₂. By adding 0.5% trypsin to the cultures and incubating them at 37°C with 5% CO₂, cellular suspensions were produced.

Morphological of apoptotic cells:

With a few minor adjustments, the approach²² was used to observe the morphological alterations of apoptotic cells. Briefly, in 60 mm dishes, 1 X 10⁶ cells were cultured for 48 hours with or without pure GEF at 48 micrograms per mL, Nano formulation at 8 micrograms per mL, and 4 micrograms per ml. After removing the medium, cells were given a PBS wash. A phase contrast inverted microscope (Leica DMI 3000B, Germany) was used to investigate the morphological alterations of the apoptotic cells.

MTT Assay:

The assay has been performed as per the method described earlier²³. Briefly, cells were seeded into 96 well plates with 15000 cells per well. The plates were then incubated overnight at 5% CO₂ at 37°C for the cells to attach. The next day, the media were replaced with fresh medium containing drugs with different concentrations and a drug-free control in triplicates. Followed by 48 hours of incubation, the plates were added with MTT (Sigma Chemical Co., St. Louis, MO) dyes and again incubated the plates for 4 more hours. The formazan crystals formed then were dissolved in DMSO and the purple colour formed was read at 570 nm using ELIZA reader.

DNA extraction and Electrophoresis:

Genomic DNA was isolated from cells previously described by²⁴. Briefly, approximately 1 × 10⁶ cells were plated and treated with pure drug (GEF) and nano-formulation (Nano-GEF) doses at specified doses. Treated cells were harvested by trypsin and washed with Dulbecco`s Phosphate Buffered Saline (DPBS), from this isolated cell, DNA was isolated by DNA isolation (Qiagen) kit according to manufacturer protocol. The concentration of DNA was determined by nanodrop at 260 nm. Then DNA was analyzed electrophoretically in 2% agarose gels containing 0.1 μg/ml ethidium bromide.

Real-time PCR:

RNA was extracted as per Aurum^TMMini Kit instructions (Bio-Rad). The RNA concentration was estimated by Nanodrop Spectrophotometer (Thermo Scientific). RNA purity was evaluated by the ratio of absorbance at 260/280nm. Isolated mRNA was used to synthesize cDNA from "cDNA Reverse Transcription Kit²⁵. The cDNA templates were used to run qRT-PCR (CFX96, Bio-Rad) by applying SYBR green master mix (Applied Biosystems). The sequences of the primers IL-6, IL-6R, gp130, NF-kB, TNF-alpha, Bcl-2, Bax, JAK, and STAT were attained from Macrogen Inc. (Korea), reaction temperature and cycle duration were as per manufacturer procedure. The GADPH expression is considered as a reference standard while gene expression is estimated as the 2^^ΔΔ CT method²⁶.

RESULTS AND DISCUSSION:

The therapeutic goal is achieved by a variety of factors. One of the most important characteristics of nanoparticles is their size. It is studied to be a main factor in accomplishing transport through biological barriers. Smaller-sized particles achieve more penetration than larger-sized particles²⁷. A narrow size distribution is indicated by a lower value for PDI. Surface charge on nanoparticles predicts colloidal dispersion stability. The zeta potential value is thought to represent a measure of surface charge. The formulations are electrostatically stabilized with a zeta potential value of (>20)²¹. As a result, the prepared Nano-GEF is considered to be a physically stable nanosystem.

Figure 1: A - the size distribution of Nano-GEF (the size and PdI were found to be 92.72 d.nm and 0.136 respectively, narrow size distribution as PdI< 0.5), B - TEM image, C - the zeta potential (-45.4 mV) (n = 1).

Morphology of apoptotic cells:

After the cells were treated with the formulations, a conventional inverted microscope was used to observe the morphological changes that occurred in the cells. After that, the cells were compared with cells that had not been subjected to any treatment. Notably, the treated cells had notable deviations from the control cells. In contrast to the untreated cells shown in (Figure 2A.), which were observed at 48 h post-treatment, treated cells exhibited several morphological changes that were detectable at this 20x magnification level (Figure 2B-D). provides a visual representation of these modifications. Cells that had been treated exhibited characteristics such as blebbing of the cell membrane, more significant inhibition of growth, and shrinking of the cells. On the other hand, cells that had not been treated continued to cling to one another during the entire incubation period. During the morphological studies, we also determined that Nano-GEF has more cytotoxic activity as compared to the pure drug, these pattern of results is previously well reported²⁸.

Figure 2: Phase contrast microscopical pictures of cell morphology after treatments (A) Control, (B) Pure GEF @ IC50 dose, 48 microgram per mL (C) Nano formulation (Nano-GEF) @ IC50 dose, 8 microgram per mL (D) Nano-formulation (Nano-GEF) @ half of IC50 dose, 4 microgram per mL.

MTT assay:

Both nano-formulation (Nano-GEF) and pure drug (GEF) were examined with MTT assay. The assay exhibits that the nano-formulation was significantly lower IC50 compared to the pure drug. The IC50 of the Pure GEF was 48 micrograms per mL, whereas the nano-formulation showed IC50 of 8 micrograms per ml (Figure 3).The cytotoxicity assay (MTT) was used to determine if Nano-GEF has anticancer properties. The improved Nano-GEF’s cytotoxicity was investigated and contrasted with GEF alone. Nano-GEF indicated a 6 times increase in cytotoxicity with an IC50 of 8 ug compared to GEF alone, which displayed considerable cytotoxicity with an IC50 of 48 ug. Furthermore, the NLC blank showed no cytotoxicity throughout the study's range. The morphological investigation also supports the cytotoxicity shown in the MTT experiment. As a result, considerable dye absorption suggests that Nano-GEF shows a similar level of cytotoxicity in cancer cells even at low doses²⁹.

Figure 3: Cytotoxicity assay performed by MTT assay. Results are average of triplicate value.

DNA fragmentation:

Induced DNA degradation was observed in the agarose gel electrophoretic pattern of both the pure drug (lane-2) and the Nano-GEF (lane-3) groups. The Nano-GEF group displayed a ladder-like pattern, which is a typical marker of apoptosis. In contrast, the control cell displayed compact DNA, which indicated that the cancerous cell did not exhibit any fragmentation (Figure 4).

Figure 4: DNA fragmentation. Control lane (left) represent solid and detectable DNA banned while pure drug lane (middle) showed some smearing pattern. Nano-GEF (Nano drug) lane (right) showed significant DNA fragmentation.

Real-Time RT-PCR:

Amplified mRNA expression of IL-6, IL-6R, gp-139, JAK-STAT, TNF-α, and Bcl-2 were all observed in control cells after treatment with the pure drug and Nano-GEF showed a reduction in the amplification gene expression in Nano-GEF as compared to the pure drug (Figure 5). However, the gene expression of BAX is increased more with Nano-GEF as compared with the pure drug (GEF) and control (Figure 6). It has been authorized to use GEF, a tyrosine kinase inhibitor (TKI) for the EGFR, to treat non-small cell lung malignancies (NSCLCs) with EGFR mutations^{30, 31}. The RAF - MAPK, PI3K-Akt, and JAK-STAT pathways are a few intracellular signaling cascades that are activated by EGFR phosphorylation and are involved in cellular proliferation, anti-apoptosis, angiogenesis activation, and cancer metastasis³². In this work, we assessed the anticancer effect of Nano-GEF and discovered that it reduced the expression of IL-6, which in turn reduced the expression of all downstream pathways proteins accountable for the genesis, development, and metastasis of cancer. Nano-GEF exerted antitumor effects by targeting IL-6, IL-6R, gp130, JAK, STAT3, and TNF-α in tumors. Nano-GEF may be a strategy for the treatment of CRC through modulation of the tumor microenvironment.

Figure 5: Relative mRNA Expression of IL-6, its Receptors and TNF-α. Data indicated as the mean ± SE. *p<0.05; **p<0.01 compared with control group; ^##p<0.01and ^###p<0.001, related to the Nano-GEF group (PD = Pure Drug, ND = Nano-Drug, ns = not significant)

Figure 6. Relative mRNA Expression of JAK, STAT 3, Bcl-2 and Bax. Values denote the mean ± SE. *p<0.05; **p<0.01 compared with control group; ^###p<0.001 related to the Nano-GEF(PD = Pure Drug, ND = Nano-Drug)

T-lymphocytes, fibroblasts, and monocytes are only a few of the typical cell types that generate interleukin (IL-6), a multifunctional cytokine. This cytokine acts via a membrane receptor complex made up of glycoprotein 130 (gp130) and IL-6 receptor a (IL-6Ra). To start the signal transduction process, IL-6 first attaches to IL-6Ra, which is unable to do so. This complex then draws gp130 molecules, which dimerize to provide the intracellular signal³³. In addition to its immunological function, IL-6 also promotes cell growth in organs including bone, the testis (spermatogenesis), the skin, and the neurological system^{34, 35}. Numerous malignancies, including melanoma, renal cell carcinoma, Kaposi's sarcoma, ovarian carcinoma, lymphoma and leukemia, multiple myeloma, and prostatic carcinoma, have been demonstrated to be stimulated by IL-6. When IL-6 or its receptors are blocked in this second tumor, chemotherapy is more effective and the tumor regresses³⁶. In our present study, we found a similar pattern of results in cytokine and receptors, showing the anticancer activity of Nano-GEF more than the pure drug.

IL-6-dependent STAT3 signaling is a crucial promoter of CRC cell survival and proliferation. Subsequently, the activation of IL-6 receptors further stimulates the signal-transduction pathways, including the signal pathway of JAK-STATs³⁷. In our work, we elucidated that the expression of JAK-STAT proteins was reduced upon Nano-GEF treatment, which seems to be according to the previous study³⁸.

Meanwhile, the activated STAT3 translocated inside the nucleus binds the STAT binding site on the promoter and starts the transcription of genes responsible for the cytokines production. Heightened inflammation in CRC, has been demonstrated to aid tumor growth³⁹. Important mediators that connect inflammation and cancer include interleukin-6 and TNF-a^40,41. The majority of cancer development processes, including tumor initiation, proliferation, migration, and angiogenesis, are influenced by IL-6 and TNF-α in CRC⁴². Patients with colorectal cancer had higher levels of TNF- α and IL-6 in their tumor tissues and sera, and these cytokines were linked to worse overall survival, metastasis, and greater tumor burden⁴³. Patients with high levels of IL-6 had lower survival times than those with low levels of IL-6 expression⁴⁴. In this work, we determined that the expression of TNF- α, IL-6 is reduced in cancer cells upon treatment with the GEF. The level of expressions is significantly reduced in the Nano-GEF treatment as compared to pure drug as it is also reported in earlier studies⁴².

Recent research on gastric cancer and multiple myeloma cells suggests that IL-6 inhibits apoptosis via controlling the expression of bcl-2 family gene products⁴⁵. The bcl-2 family gene products, which include the inhibitors (bcl-2, mcl-1, or bcl-xl) and promoters (bax, bad, or bak), play a significant role in the control of apoptosis⁴⁶. The expression of Bax and Bcl-2 in human breast cancer has been discussed by several writers⁴⁷. The main factor that determines whether or not apoptosis is induced or inhibited appears to be the bcl-2 to bax ratio. One of the most significant characteristics of cancer is thought to be the breakdown of this equilibrium⁴⁸. In the present work, we found that the expression of Bcl₂was decreased while the level of Bax was increased by nano-formulation (Nano-GEF) as compared with the pure drug (GEF), while the increased ratio of Bax/Bcl₂ is beneficial in cancer treatment.

CONCLUSION:

We conclude that the increased cell proliferation seen in cancer may be connected to the elevated expression of IL-6 and its receptors in CRC. The proliferation/apoptosis balance may be changed toward the growth of cancerous cells by IL-6 by boosting IL-6R expression. Upon treatment with the Nano-GEF, the reduction in the expression of all the pathways responsible for cancer progression. So, we can assume from our study that Nano-GEF could be an effective treatment method for colorectal cancer.

ACKNOWLEDGMENTS:

The authors would like to express their gratitude to the Deanship of Graduate Studies and Scientific Research, Jazan University, Saudi Arabia, for providing funds through project number: (RG24-M025).

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

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Received on 11.04.2024 Modified on 05.07.2024

Research J. Pharm. and Tech 2024; 17(10):4968-4974.

DOI: 10.52711/0974-360X.2024.00764