Nerolidol, bioactive compound suppress growth of HCT-116 colorectal cancer cells through cell cycle arrest and induction of apoptosis

Abstract


Introduction
Cancer is becoming one of the most serious diseases affecting human health.Colon cancer is the widespread and serious type of cancer, with high rates of occurrence and fatality rates even in developed countries [1,2].Colon cancer is the third most deadly cancer globally, with a projected 900,000 mortalities per year in both men and women by the year 2020 [3].This places a signi cant burden on the healthcare system.More than 50% of colon cancer patients are cured by the surgical removal along with chemotherapy, however, 40-50% of these patients are still develop a recurrence, and the chances of complete recovery is really poor.The possibility of a patient surviving this illness is increased by early identi cation and treatment, which highlights the signi cance of effective and speci c medical care in cancer management [4].Currently, chemotherapy, radiation, and surgery are the methods used to treat people with colon cancer.The ve-year survival rate of colon cancer patients remains low at 64% even though advances in treatment.[5].Therefore, patients with colon cancer need access to new and more potent anticancer drug candidates to enhance treatment results and decrease side effects.
Tumor cells speci cally modify their biological functions during cancer progression to favor rapid growth, migration, and resistance to genotoxic and metabolic stressors [6].Apoptosis is a genetically regulated process that eliminates damaged cells to preserve the tissue's cellular equilibrium.It is believed as a crucial part of several cellular mechanisms.Defective apoptotic signals may eventually encourage the development of cancer [7].Inhibition of apoptosis by certain pro-survival proteins is crucial to the maintenance of tumor phenotypes in order to evade the cell death enforced by oncogenic stress during tumor formation.As a result, many crucial mechanisms regulating apoptosis and cell survival are modi ed in cancer [8].To avoid apoptosis, cancer cells can develop a number of different defense mechanisms [9].The apoptosis induction in tumor cells is a major strategy used by anticancer therapies like radiation, chemotherapy, and targeted therapies [10].
The resistance to chemotherapy is a signi cant issue because it is one of the rst-line treatment options for treating aggressive types of colon cancer [11].The several chemotherapy drugs are being used to treat colon cancer and decrease death rates, however, the clinical usage of these medications are restricted and successful recovery of patients are poor due to the long-term side effects [12].Therefore, improved therapy options are required for the effective treatment of colon cancer.Natural substances are being investigated as talented cancer therapies with promise.In reality, 87% of all identi ed human diseases are being treated with natural substances, and 74% of the most effective life-saving medications are made with active components obtained from plants [13].Hence, the development of drug substitutes from natural sources may be a bene cial strategy for treating colon cancer.
Nerolidol, a sesquiterpene alcohol, occurs in the many essential oils of different plants [14].The antiin ammatory, antibacterial, anti-neurodegenerative, and anticancer properties of nerolidol are well documented [15,16].Furthermore, nerolidol has been reported to demonstrate neuroprotective [17], hepatoprotective [18], cardioprotective [19], genoprotective [20], and anti-nociceptive [21] properties.It can inhibit HepG2 hepatocellular carcinoma cell growth [22] and improve the cytotoxicity property of doxorubicin on breast cancer [23].However, its anti-colon cancer properties have not yet been revealed.Therefore, the current research was carried out to demonstrate the salutary roles of nerolidol in the proliferation and cyctotoxic mechanisms of colon cancer HCT-116 cells.

Cell Collection And Maintenance
The colon cancer HCT-116 cells were obtained from ATCC, USA, and grown on DMEM enriched with 10% FBS in a CO2 (5%) incubator.Cells were gathered and used for the subsequent assays once they reached 80% con uency.

Mtt Cytotoxicity Assay
By using the MTT assay, the impact of nerolidol on the HCT-116 cell viability was assessed.On a 96-well plate, cells were cultured at a population of 5 × 10 3 for 24 hours.Following that, cells received nerolidol (5,10,25,50,75, & 100 µM) treatment for 24 hours.Following the treatment, each well received a mixture of 20 µl of MTT reagent and 100 µl of DMEM, which was then maintained for 4 h.After dissolving the developed formazan stones with DMSO (100 µl), the absorbance was measured at 570 nm using ELISA reader.

Dual Staining
By using dual staining, the extent of apoptosis in nerolidol-treated HCT-116 cells was investigated.Cells were grown on a 24-well plate at a population of 5 × 10 5 cells/well and then 25 and 50 µM of nerolidol was treated for 24 h.To evaluate apoptosis in HCT-116 cells, 100 µg/ml of AO/EB stains were added to each well and incubated for 5 minutes at dark.The intensity of the generated uorescence was then assessed under a uorescent microscope.

Dcfh-da Staining
By using the DCFH-DA staining method, the ROS production in both control and nerolidol-treated HCT-116 cells was evaluated.The 24-well plate was used to culture the HCT-116 cells at a population of 5×10 5 cells per well and then nerolidol at 25 and 50 µM concentrations were treated for 24 h.After that, 10 µl of DCFH-DA dye was mixed into each well and then incubated for 1 hr at 37°C.The uorescent microscope was utilized to investigate the intensity of the developed uorescence, which correlates to the level of ROS formation.

Dapi Staining
Using DAPI staining, the altered apoptotic nuclear morphology of HCT-116 cells caused by nerolidol was examined.The HCT-116 cells were added to the 24-well plate and then nerolidol at dosages of 25 and 50 µM were treated for 24 h.Then, cells were xed using paraformaldehyde (4%) for 30 minutes and then cells were stained with DAPI (200 µg/mL) for 15 minutes.The changes to the chromatin caused by nerolidol in the HCT-116 cells were then examined using uorescent microscope.

Cell Cycle Analysis
The control and nerolidol-treated HCT-116 cells were collected at a population of 5 × 10 6 cells/mL, treated with 70% ethanol, and then incubated for 12 hours.Following cell washing, staining solution (300 µl) including PI (100 µl), proteinase inhibitor (0.08 mg/ml), and RNase (0.5 mg/ml) were mixed, and the solution was incubated for 30 minutes.The ow cytometry was utilized to determine the DNA-related PI uorescence.The MultiCycle software was used to calculate the proportions of nuclei in each cell cycle stages, including G1, S, and G2/M.(Phoenix Flow Systems, USA).WinMDI 2.9 software was used to detect the percentage of sub-diploid cells (apoptotic cells).

Statistical analysis
Values are revealed as the mean ± SD of three different assays after the results were examined using the GraphPad Prism software.One-way ANOVA and DMRT were performed to investigate changes in the values of the treatment groups, and a signi cance at p < 0.05 was used.

Effect of nerolidol on the viability of HCT-116 cells
Figure 1 demonstrates the viability of control and nerolidol-treated HCT-116 cells.Our ndings showed that, when compared to the control, the nerolidol treatment at 5-100 µM concentrations substantially reduced the viability of HCT-116 cells.The IC50 level of nerolidol was found at 25 µM, which inhibits 50% of the viability.As a result, the 25 and 50 µM of nerolidol were chosen for the subsequent analyses as IC50 and high dose treatments.

Effect Of Nerolidol On The Ros Level In The Hct-116 Cells
The impact of nerolidol on the generation of ROS in HCT-116 cells is revealed in Fig. 3.The control cells exhibited the low green uorescence, whereas the HCT-116 cells exposed to 25 and 50 µM nerolidol demonstrated a strong green uorescence, which indicates that the nerolidol treatment signi cantly enhanced ROS accumulation in the HCT-116 cells.These outcomes demonstrate that nerolidol facilitate oxidative stress-mediated damage to the HCT-116 cells by increasing the generation of intracellular ROS.
Effect Of Nerolidol On The Apoptotic Cell Nuclear Morphology In The Hct-116 Cells DAPI staining was done to evaluate the impact of nerolidol on the apoptotic cell nucleus in HCT-116 cells and the outcomes are revealed in Fig. 3. DAPI staining results showed that the 25 and 50 µM nerolidoltreated HCT-116 cells had distinct changes in nuclear morphology such as damages, shrinkage, and formation of apoptotic bodies, which reveals that nerolidol increases apoptosis in HCT-116 cells (Fig. 3).
Effect Of Nerolidol On The Apoptotic Cell Death In The Hct-116 Cells Therefore, it was evident that nerolidol treatment dramatically increased the apoptosis in the HCT-116 cells.

Effect Of Nerolidol On The Cell Cycle Arrest In The Hct-116 Cells
Using ow cytometry study, the cell cycle phases in the control and nerolidol-exposed HCT-116 cells was studied (Fig. 5).The nerolidol at the dosages of 25 and 50 µM treated HCT-116 cells exhibited a more percentage of cells in the G0/G1 phase.Also, the percentage of cells in the G2/M phase was lower in the HCT-116 cells after they were exposed to the 25 and 50 µM of nerolidol.This showed that the nerolidol treatment inhibited the cell cycle at G0/G1 phases (Fig. 5).

Discussion
Colon cancer remains the major cause of cancer-associated mortalities in both men and women globally [24,25].Furthermore, resistance to therapies made it more di cult to treat colon cancer [26].Therefore, there is a need to look for novel, effective, and low-toxic alternatives that could effectively prevent and/or inhibit the progression of colon cancer.In the current work, the anticancer potential of nerolidol against the HCT-116 cells were studied and the results were demonstrated the signi cant bene cial activities of the nerolidol.The tumor cells have a strong ability to replicate themselves and metastasize to other body parts.The uncontrolled and excessive cell proliferation causes the development of cancerous tissues, which have a propensity to metastasize to other body parts [27].In order to demonstrate the anticancer effects of nerolidol, we attempted to discover the inhibitory potential of nerolidol on the proliferation of colon cancer HCT-116 cells using the MTT assay.The ndings reveal that nerolidol treatment substantially obstructs the HCT-116 cell viability.The current ndings were supported by previous research done by Lee et al. [28], who found that podophyllatoxin effectively decreased the HCT-116 cell viability.
ROS are highly reactive substances produced by mitochondria as a metabolic byproduct, which aid in the malignant transition of normal cells into neoplastic precursors [29].Additionally, ROS are crucial intracellular signaling mediators under normal circumstances, but an abnormal gathering causes oxidative stress in cells and encourages apoptosis [30].In order to support their fast proliferation, cancer cells produce more ROS than normal cells do.However, excessive ROS levels can cause tumor cell death by the number of pathways [31].Cancer cells are more susceptible to medicines that increase ROSmediated oxidative stress.In cancer treatment, the stimulation of higher ROS accumulation in the tumor cells is an effective approach [32].It was evident that several bioactive substances with anti-cancer potential raise ROS level, triggering oxidative stress-mediated apoptosis in tumor cells [33].In the current work, the ndings of DCFH-DA staining demonstrated that nerolidol effectively augmented ROS level in HCT-116 cells, which may facilitate the oxidative stress mediated apoptotic cell death.The previous study done by Lin et al. [34] found that ferruginol effectively increased the ROS level in HCT-116 cells, thereby facilitating oxidative stress-mediated cell injury.These outcomes are supported the results of the current research work.
In addition to uncontrolled cell proliferation, defective apoptosis is also a major contributing factor in tumor growth.Apoptosis is a crucial defense mechanism against the progression of tumors.Neoplastic cells undergo oncogenic transformation when genetic and epigenetic changes cause them to become resistant to apoptosis.In tumorigenesis, the defective apoptosis regulation causes an extended life span of tumor cells, growth under stress, tumor angiogenesis, and metastasis.It also adds to therapeutic resistance in tumor cells [35].The pathophysiology of apoptosis is multifaceted and incorporates extrinsic and intrinsic signal transduction [36].Tumor cells produce several mechanisms to prevent apoptotic cell death, most notably the overexpression of anti-apoptotic molecules.DNA injury in precancerous lesions can cause apoptosis, which eliminates potentially hazardous cells and prevents tumor growth.Contrarily, apoptotic dysfunction can cause uncontrolled cell proliferation, tumor growth, and tumor resistance to therapies [37].Therefore, apoptosis stimulation in cancer cells is considered a most promising approach to obstruct tumor progression.In this study, the results of dual staining exhibited that nerolidol treatment effectively stimulated apoptosis in HCT-116 cells, which evidences that nerolidol can inhibit colon cancer growth by triggering apoptotic cell death.This observation is in agreement with the previous research done by Xiang et al. [38], who found that curcumin triggered apoptosis in HCT-116 cells, which was analyzed by dual staining.
Some apparent morphological abnormalities that occur during apoptosis can be considered indicators of the beginning of the apoptotic process.In summary, apoptosis begins with chromatin condensation in the nucleus, progresses through nuclear fragmentation, and nally results in the formation of apoptotic bodies [39,40].The results of DAPI staining also demonstrated increased apoptotic cell nuclear morphology, which is evident by increased damaged cell morphology, nuclear fragmentation, and apoptotic bodies in the nerolidol-exposed HCT-116 cells.Hence, it was clear that nerolidol can be useful to treat colon cancer by encouraging apoptosis.Our present results were supported by the previous research work done by the Dangroo et al. [41].
In addition to cell toxicity and apoptosis induction, the impact of nerolidol on the cell cycle arrest in the HCT-116 cells was also investigated in this study.It is crucial to understand that there are various stages in the cell cycle, including gap 1 (G 1), gap 2 (G 2), and mitosis (M) [42].A variety of regulatory molecules are involved in the intricate process of the cell cycle, which controls the growth of cancer cells [43].The checkpoints of cell cycle are commonly stimulated due to the DNA damage and replication stress.Each checkpoint in normal cell division is strictly controlled, and any disruption of those checkpoints causes cells to divide rapidly and uncontrollably [44].A possible target for cancer therapies is the dysregulation of the cell cycle, which promotes the development of tumor [45].It was reported that some naturally occurring bioactive compounds, such as hydroxy-g-sanshool, inhibit colon cancer cell growth by inducing the sub-G1 population [46].According to the current ndings, we found that, compared to control cells, HCT-116 cells exposed to nerolidol had a higher sub-G1 population.Therefore, it was clear that the nerolidol caused the inhibition of cell cycle in the G0/G1 phase and reduced HCT-116 cell growth.A recent studies done by the Zhaojun et al. [47] and Kwon et al.[48] found the inhibition of cell cycle at G0/G1 phase in the HCT-116 cells using bioactive compounds, which supports the results of the present study.

Conclusion
In conclusion, our results show that nerolidol can inhibit viability and accelerate apoptotic cell death in HCT-116 cells by elevating intracellular ROS accumulation and inducing cell cycle arrest.In light of these ndings, nerolidol is recommended as a talented and salutary candidate to treat colon cancer in the future.More study will be compulsory in the future to fully comprehend the different molecular pathways that make nerolidol effective against colon cancer as an anticancer drug.

Figure 4
Figure 4 depicts how nerolidol in uenced the apoptosis in HCT-116 cells, which was investigated by dual staining.The HCT-116 cells exposed to 25 and 50 µM nerolidol showed a bright orange/yellow uorescence with damaged cell morphology, which are in contrast to the untreated control cells.

Figure 1 Effect
Figure 1

Figure 2 Effect
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Figure 3 Effect
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Figure 4 Effect
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Figure 5 Effect
Figure 5