Evaluation of the Effects of Curcumin and its Two Nanoforms on the Cancerous and Normal Cell Lines in the Presence of Radiation

doi:10.21203/rs.3.rs-2224966/v1

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Evaluation of the Effects of Curcumin and its Two Nanoforms on the Cancerous and Normal Cell Lines in the Presence of Radiation

https://doi.org/10.21203/rs.3.rs-2224966/v1

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The medicinal properties of herbs attract the public and the medical community’s attention. Turmeric is considered a natural substance with many health effects, and curcumin, as effective component of turmeric, is known to have several usefulness for human health. The dual effect of the curcumin in the presence of radiation as a radiosensitizer and radioprotector for tumor and normal cells, respectively, increases treatment efficacy. In this study we have investigated the effect of X-ray radiation (6 MV) and bulk curcumin, nano micelles, and nanoniosome of curcumin on two colorectal cancer cell lines and human fibroblast cells using MTT, colonogenic assay, and annexin-PI test. Based on the results, curcumin and the two formulations used decreased the viability of both cell lines, however the final results depend on the curcumin concentration. The results of the dose-response curve displayed that the survival fraction in the presence of nanomicelles had a dual function on cancerous and normal cells due to a radiosensitivity and radioprotective effects on cancerous and normal cells, respectively. However, these effects were not shown in the presence of nanoniosome of curcumin. Curcumin in all 3 forms increased apoptotic cells in both cell lines without any radiation. However, the apoptotic cells increased in exposed cancerous cells for all forms of curcumin, the apoptotic HFF cells decreased after radiation in presence of them. In conclusion, curcumin is considered a radiosensitizer in tumor cells, and its radioprotection properties need more studies.

Curcumin

Nanomicelle

Nanoniosome

Radiation

Curcumin, as a yellowish polyphenolic pigment is extracted from the rhizome of turmeric (Curcuma longa plant) [1], is mostly used in cooking and food industry in Traditional Chinese Medicine (TCH) and Iran. It is also generally recognized as safe (GRAS) by the United States Food and Drug Administration (FDA) therefore, its use in clinical practice is not restrained [2].

Curcumin has been mostly studied due to the promising biological and pharmacological effects, and many studies have revealed modulatory effects of curcuminoids on many biomolecules. Furthermore some researchers have reported that, the curcumin plays a vital role in the pathways regulating inflammation, oxidative stress, immune response, and cellular proliferation, as well as homeostasis [2].

The free radical scavenging, enzyme modulating, antioxidant, anti-inflammatory, anti-arthritic, anticarcinogenic, cardioprotective, and hepatoprotective possessions are known as natural polyphenols properties, particularly in curcumin [3].

Curcumin was first used as a radioprotector in 1990, but radioprotective and radiosensitive effects on normal and cancerous cells respectively were known later. Curcumin could be considered an ideal radioprotector since curcumin, as a natural antioxidant, can capture and remove the free radicals produced damage and also enhances the activity of existing superoxide dismutase in cells. Additionally, curcumin affects signaling pathways, such as nuclear factor (NF)-KB as a transcription factor with a significant effect on the induction of cytokine expression in the inflammatory response, including the radiation-induced cascade of inflammatory responses. Further, curcumin decreases the release of inflammatory factors, including monocyte chemotactic protein (MCP)-1, interleukin (IL)-1, tumor necrosis factor (TNF)-α, protein kinases, apoptosis, and DNA repair pathways. Therefore, utilization of curcumin may have a synergistic effect in the treatment of cancer [1].

On the other hand, many issues are incorporated in adoption of curcumin, such as the low aqueous solubility, instability at physiological pH, rapid metabolism, and clearance; the poor pharmacokinetic profile of curcumin is the major drawback to use in the therapeutic application [4, 5].

The use of curcumin has been controversial in clinical application, in spite of its role in multiple preclinical models, poor oral absorption limits the systemic bioavailability of curcumin, leading to prescription extremely high doses for clinical efficacy [2, 6].

In some studies researchers have tried to improve the pharmacokinetic characteristic of curcuminoids, oral biological availability, and pharmacological effects [2]. Curcumin nanoformulations are considered a novel strategy to enhance the bioavailability and properties of curcumin [7]. Nanoparticles-based delivery systems, such as liposomes or micelles, could solve these problems [5]

Drugs and chemicals

In this study, the HT-29 human colon cancer cell line and human foreskin fibroblasts (HFF) were purchased from the Pasteur Institute, Tehran, Iran. Fetal bovine serum (FBS), DMEM, and RPMI 1640 were used as culture medium, these were supplied by Gibco (USA). Furthermore, Dimethylsulfoxide (DMSO), MTT (3-[4, 5 dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide), trypan blue, penicillin, streptomycin and trypsin–EDTA and PBS buffer were obtained from Sigma (St. Louis, MO, USA); Geimsa has been purchased from Merck (Germany).

Cells Culture

HT-29 cells were continuously grown as a monolayer in RPMI 1640 with 10% FBS, penicillin (100 U/ml), and streptomycin (100g/ml) in an incubator humidified atmosphere containing 5% CO2 at 37°C in the 75 cm ³ flasks (SPL). HFF cells were cultured in DMEM, including 10% FBS at similar conditions; a culture medium was replaced by fresh medium every 3 days.

Drugs

Nanomicelle containing curcuminoids was registered as SinaCurcuminR obtained from Exir Nano Sina Company, Tehran, Iran (IRC: 1228225765) [2]. Curcumin-loaded niosome nanoparticles were obtained from Food Science and Technology Research Institute, Mashhad, Iran. These nanoparticles were prepared using the thin-film hydration method that curcumin was encapsulated in the shell of niosome nanoparticles and their characteristics were described previously[8].

Cells Toxicity Of Drugs

The cells with 80% confluency were detached with trypsin–EDTA, centrifuged at 1200 rpm for 5 min and counted using trypan- blue with Neubauer slide to ensure viability. The cell suspensions (5 × 10⁵ cells/ml) were seeded in a 96-well plate, incubated for 24 h, and treated with different concentrations of 3 drugs separately (0-200 µM) for 24 h. The cells were then washed with PBS to remove the remaining drugs. The percentage of dead cells were immediately assessed and compared with untreated cells by MTT assay.

Mtt Assay

The MTT-tetrazolium assay determined the viability of the cells. The yellow tetrazolium compound changed to purple formazan in live cells' mitochondria. After removing the cell culture medium, 100 µl of fresh culture medium without FBS and 10 µl of MTT (5 mg/ml in PBS) were added to each well and incubated for 24 h to proceed with the assay. The media was removed from the wells, and the cells were lysed with 200 µL of DMSO. The samples' optical density (OD) was measured at 570 nm by an ELISA reader (Stat Fax-2100 Awareness, Mountain View, CA, USA), and relative survival was calculated as the OD of the treated sample divided by the OD of the control group. Eight replicates were used for each group, and the experiments were repeated at least three times.

Colony Formation Assay

In this part, cells were treated with radiation and selected concentration of all three forms of curcumin, seeded in 6 well plates at very low concentrations (0.1-1K), and incubated. After 24h, the plates were exposed to radiation (0, 2, 4, and 6 Gy), were left in the incubator for 2 to 3 weeks, fixed with fixation solution, and stained with Geimsa (enough stain to cover the entire plate). All colonies with larger than 50 cells were counted with the ImageJ (version 1.41o, Java 1.6.0_10,Wayen Rasband, US National Institutes of Health, Bethesda, MD, USA). The number of colonies from plates with or without prior curcumin treatment were compared; every 3 plates received similar treatment. A standard deviation was also calculated.

The survival fraction (SF) is equal to [9]:

SF = number of colonies formed after treatment/ number of cells seeded*PE

Where PE (Plating efficiency) is the ratio of the number of colonies to the number of cells seeded: PE = number of colonies formed /number of cells seeded *100%

Determination Of Cell Death By Annexin V/pi Staining

The quantities of apoptosis and necrosis induced by curcumin annd nanoparticles were measured by the Annexin V/PI staining kit (Mahboub Bioresearch Company, MBR, Tehran, Iran). The cells were seeded at 1 × 10⁶ cells per well on a 6-well plate and treated with curcumin and nanoparticles (10 µM) for 24 h. Cells were then washed with 200 µl of 1× binding buffer and centrifuged at 1200rpm for 5 min. Subsequently, 10 µl of AnnexinVFITC reagent were added to the cells and incubated for 15 min in dark at room temperature. Then the samples were incubated with 3 µl PI reagent for 5 min, and the free reagent were remove by PBS. Next, the final volume was set at 500 µl with PBS. The flow cytometer immediately quantified the number of viable, early, late apoptotic, and necrotic cells (BD FACSCALIBUR™ Becton Dickinson, USA). The results were analyzed by FlowJo V10 software (Flowjo, USA).

Statistical analysis

The data were analyzed by the Statistical Package for Social Sciences (SPSS Inc., version 25, Chicago, IL, USA); Kolmogorov–Smirnov assessed the normality of the data. One-way ANOVA followed by Tukey’s multiple comparison tests was also used to compare all groups with statistical significance (p-value < 0.05). GraphPad Prism software (version 8.4.3; GraphPad Software Inc., La Jolla, CA) and Microsoft Excel (version 2019) were used for all figures.

The viability of the cells in the presence of three forms of curcumin

The viability curve of HT-29 and HFF cells are showed in Fig. 1. The viability percentage of the cells significantly decreased with increasing curcumin concentration in all forms. The half-maximal inhibitory concentration (IC50) of curcumin, nanomicell, and nanoneosome obtained were approximately 55, 65, and 160 µM for HFF and 20, 38, and 135 for HT-29 cells.

The Dose-response Curve Of The Cells In The Presence Of Nanocurcumins

Figure 2 shows dose-response curve of HT-29 and HFF cells obtained by colonogenic assay. A dual effect of nanomicelle was demonstrated in the cells. The survival fraction of the cancerous and normal cells decreased and increased after radiation, respectively. The behavior of nanoniosome of curcumin was similar to the control. the difference that is shown some radioprotection effect in HT-29 cells (Fig. 2).

Cell Apoptosis In The Presence Of Three Forms Of Curcumin

The cells were treated with various forms of curcumin and apoptosis parameters were evaluated by using Annexin V-FITC binding analysis and PI staining to quantify the cell death, increasing from necrosis and apoptosis. As shown in Fig. 4, the percentage of apoptotic cells was 10–12% in the control group. In both cancerous and normal cells, the number of Annexin V-FITC-/ PI-positive cells were increased following treatment with all forms of curcumin. The apoptosis parameters were increased by more than 50% in HT-29 cells in comparison with the control group. However, the HFF cells increased by 5 folds in the control group in the absence of radiation. After applying curcumin, nanomicells, and nanoniosome of curcumin, the percentage of HT-29 cells in the apoptotic stages was about 21.7, 18.1, and 16.9%, and in HFF cells, 47.6, 64, and 49.4%, respectively (Fig. 3). However, these parameters were markedly increased in cancerous cells after radiation; in normal fibroblast cells, apoptotic parameters were decreased, dependent on the radiation dose. Flow cytometry analysis revealed that the percentage of early and late apoptotic cells following administration of curcumin, nanomicells, and nanoniosome of curcumin at 2Gy was 36, 27.6, and 31.2% in HT-29 cells and 44.4, 50.7, and 36.5% in HFF cells, sequentially. When HT-29 cells exposure was increased from 2 Gy to 4Gy, the percentage of apoptotic cells increased only up to 10%. On the contrary, when radiation dose delivered to the HFF cell was risen up, the number of apoptotic cells declined, showing a late apoptotic parameter (Fig. 3).

Based on our results in this study, normal cells demonstrated a higher sensitivity to all three forms of curcumin than cancerous cells. The presence of 3 forms of curcumin could increase and decrease apoptosis in cancerous and normal cells, respectively.

Curcumin, the active ingredient of turmeric, has been used in traditional medicine in Asian countries, such as China and India, since 2000. Additionally, unique features of curcumin have also been considered a curative or sedative drug for some diseases, such as diabetes, skin injuries, excisional wounds some types of cancers. This compound increases radiation sensitivity in tumors and exhibits protective properties against many complications caused by radiation. This dual function is essential in radiotherapy due to increase in the efficiency of treatment based on increasing tumor sensitivity and reducing side effects. The present study investigates the dual function of curcumin and two of its nanoparticle forms.

The radiosensitivity of tumor cells clearly was increased in the presence of curcumin, which is consistent with the results of previous studies.

Khafif et al. introduced curcumin as a new radiosensitizer in squamous cell carcinoma and suggested that arrest at the S/G2M phases of the cell cycle can have a radio-sensitizing impact on the cells treated by curcumin [10]. Based on the study by Kunnumakkara et al., curcumin suppressed NF-kB and regulated gene products in colorectal cancer. Therefore it is potentiates as an antitumor drug of radiation therapy by leading to inhibition of proliferation and angiogenesis [11]. So, Sandur et al. emphasized that transient inducible NF-kB activation provides a prosurvival response to radiation, leading to the development of radioresistance in colorectal cancer. Curcumin blocks the signaling pathway and potentiates the antitumor effects of radiation therapy [12]. Qiao et al. have showed curcumin's important role in radiotherapy by inhibiting the PI3K/AKT-dependent NF-κB pathway and enhancing radiation-induced apoptosis in Human Burkitt’s Lymphoma [13]. Schwarz et al. investigated the effect of curcumin on human pancreatic cancer cell lines and showed a significant enhancement of radiation-induced apoptosis, DNA-double-strand breaks, and G2/M-arrest after curcumin treatment in both cell lines [14].

Curcumin in covers of nanomaterials, such as micelles, increases solubility, stability, and delivery of curcumin. Hatamipour et al. introduced a novel nanomicelle formulation for the delivery of curcumin as a commercial drug [2]. Although, many studies have investigated the effects of nanomicelles of curcumin on cancerous cells [5, 15], few studies have assessed the radiosensitivity of these forms of curcumin in a clinical trial on prostate cancer patients. Saadipoor et al. did not succeed to demonstrate the efficacy of nanocurcumin for concurrent therapy in prostate cancer patients undergoing RT [16]. But in our study, increasing of radiosensitivity of tumor cells was reported in presence of nanoforms of curcumin.

However, Results of the present study are in good agreement with finding of other researchers on cancerous cells, in contrast, in our work it was revealed that apoptotic parameters were decreased in the presence of curcumin and nanoform`s in normal cells.

On the other hand, in our study, the nanomicells of curcumin showed radioprotective effects on normal fibroblast cells, suggesting dual function of this form of curcumin. However, the curcumin and nanoniosome`s decreased apoptotic parameters in normal cells, it did not show the radioprotective effect due to the certain immunological response, which had an important role in the effects of curcumin or nanocurcumin on the treatment efficacy.

On the basis of literature, the radioprotection effect on normal cells in present of curcumin or nanocurcumin, were showed in vivo studies, but few workers in their in vitro studies reached to this conclusion with the emphasis on the role of immunological response [1, 6, 7, 17–19]. However, we have showed radioprotection effect in presence of nanomicells of curcumin produced by alteration of signaling pathways, suggesting that the effect of other nanoforms of curcumin examined in vivo.

In conclusion, the findings of this study have showed that curcumin and two forms of nanocurcumin could be used as radiosensitizer component but it`s radioprotective effects on normal cells needs to future study. It is better to be considered the role of immunological response.

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Editorial decision: Major Revisions Needed
07 Dec, 2022
Reviewers agreed at journal
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Editor assigned by journal
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You are reading this latest preprint version

Evaluation of the Effects of Curcumin and its Two Nanoforms on the Cancerous and Normal Cell Lines in the Presence of Radiation

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Drugs and chemicals

Cells Culture

Drugs

Cells Toxicity Of Drugs

Mtt Assay

Colony Formation Assay

Determination Of Cell Death By Annexin V/pi Staining

Statistical analysis

Results

The viability of the cells in the presence of three forms of curcumin

The Dose-response Curve Of The Cells In The Presence Of Nanocurcumins

Cell Apoptosis In The Presence Of Three Forms Of Curcumin

Discussion

Conclusion

References

Status:

Version 1