Nanoencapsulation of Cordycepin Induces Switching from Necroptosis to Apoptosis in Human Oral Cancer Cells (HSC-4) Through Inhibition of Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3) and Autophagy Modulation

Abstract

Human oral squamous carcinoma is considered the most common oral cancer; it imposes multiple oral and dental consequences as a result of oral cancer treatment. We previously found that the nanoencapsulation of cordycepin (CS) could inhibit oral cancer cells. However, the mechanism of action was not understood. The aim of this study was to investigate the signaling pathway by which CS and encapsulated nanoparticles (NPs) activate the inhibition of cancer cell growth. We demonstrated that human oral cancer (HSC-4) cells underwent necroptosis when incubated with high concentrations of CS, but not when incubated with either low concentrations of CS or encapsulated CS NPs. High concentrations of CS-induced necroptosis of HSC-4 cells, demonstrated by a reduction in apoptotic (BAX, Caspase-3, Caspase-8, and Caspase-9) and autophagic genes (LC3, Atg5, and Atg12). However, low concentrations of CS significantly induced the expression of autophagic gene LC3. Interestingly, encapsulated CS NPs induced a significant increase in apoptotic genes (P53, BAX, Caspase-3, Caspase-8, and Caspase-9), but a significant decrease in autophagic (P62, Atg5, and Atg12) and necroptotic genes (receptor-interacting serine/threonine-protein kinase 3 [RIPK3]). We also found that encapsulated CS NPs enhanced the accumulation of cellular protein and decreased secreted supernatant protein levels. Moreover, encapsulated CS NPs had higher efficacy in terms of reactive oxygen species (ROS) generation-mediated inhibition of autophagy compared to nonencapsulated CS, suggesting that nanoencapsulation of CS can switch the program of HSC-4 cell death from necroptosis to apoptosis. In conclusion, HSC-4 cells have a defense strategy against CS-induced cell apoptosis, but this problem can be solved through the use of encapsulation combined with nanotechnology.

Introduction

Human oral squamous carcinoma is one of the major head and neck cancers, ¹ and is the sixth most common cancer as it constitutes approximately 5% of human malignancies. ² The most popular choices for oral cancer treatment are surgery, radiotherapy, and chemotherapy. ³ However, conventional therapies are often unable to cure malignant tumors that exhibit resistance to radiotherapy, and new treatments are needed. ⁴ These cancer treatments may cause side effects such as oral mucositis, dry mouth, skin damage from radiation, and infections. ⁵ One therapeutic strategy is to reduce the number of cancer cells by causing apoptotic cell death. ⁶

According to past research, nanoparticles (NPs) have been used to increase the stability of bioactive compounds. There are many studies about the action of NPs in cancer inhibition; for example, NPs have been conjugated with antibodies against GRP78 to inhibit the adhesion, invasion, and metastasis of hepatocellular carcinoma and promote the delivery of 5-fluorouracil into GRP78 highly expressing human hepatocellular carcinoma cells. ⁷ In oral treatment, cisplatin-loaded NPs (NC-6004) have been shown to activate the Caspase-3 and Caspase-7 pathways to induce apoptosis in oral cancer cells. ⁸ Moreover, curcumin-loaded chitosan-coated NPs can cause apoptosis and inhibit proliferation of oral tumor cells. ⁹

Natural extracts that have anti-cancer properties are gaining more attention at present. Cordycepin (CS) extracts from Cordyceps, for instance, have the ability to inhibit many types of cancer cells. ¹⁰ However, our previous study reported that oral cancer cells could exhibit self-defense mechanisms against low concentrations of CS. ¹¹ Thus, high concentrations of CS, which were toxic and led to necrotic cell death and inflammation, were required to suppress these cells. ¹² In the same study, our group developed 3 forms of nanoencapsulation of CS, ie, CS standard-loaded cassava starch (CSNPs) and cordyceps medium-loaded CS NPs using either physical or acid treatment (CMP and CMA, respectively). Testing the ability of these new forms in oral cancer cell inhibition, we found that not only they could decrease the CS dose, but the cellular swelling observed in cell necrosis was also not present. ¹¹ Therefore, despite an unknown underlying mechanism, we hypothesized that nanoencapsulated CS could induce apoptosis in oral cancer cells instead of necrosis. These findings led to the goals of the present study, which were to investigate the self-defense mechanism of oral cancer cells against CS and examine the mechanistic pathways of encapsulated CS NP-induced inhibition of cancer cell growth.

Material and Methods

Materials

CS standard (MW, 251. 2; product No. C3394), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemical Co. () and Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (HyClone).

Cell Culture

Human oral squamous cell carcinoma (OSCC) cell lines (HSC-4) were obtained from the Department of Biology, Mahidol University, Thailand. Cells were cultured in DMEM containing 10% (v/v) FBS, 1% (v/v) nonessential amino acids, 1% (v/v) L-glutamine, and 1% (v/v) penicillin-streptomycin in a humidified incubator at 37 °C and 5% CO₂.

Fabrication of Encapsulated Nanoparticles

The preparation methods for CSNPs were performed as previously described. ¹¹ The method was composed of 3 processes; (1) extraction of starch, (2) encapsulation of CS, and (3) aqueous nanosuspension of CS-loaded CSNPs. A nanosuspension was prepared to obtain either a soluble form of CS or cordyceps medium-loaded CSNPs using either physical or acid treatment (CMP and CMA, respectively). For physical treatment, 1. 5% cassava starch powder was pre-treated with 0. 1 M NaOH for 1 h, centrifuged, and resuspended in distilled water in equal volume. Ethanol was then added to the starch solution under continuous magnetic stirring. The hydrolyzed starch was centrifuged, and the pellet washed and dried in the oven. In acid treatment, 10% cassava starch powder was mixed with 3. 16 M sulfuric acid and the mixed solution was shaken for 5 days. Then, the suspension was washed and centrifuged. The pellet was washed, the pH adjusted, and dried in the oven. Furthermore, 10% hydrolyzed starch from both treatments was mixed with 2. 5 mM CS and either 2. 5 or 5 mM cordyceps medium. Encapsulated starch was centrifuged before washing and drying the pellet. Afterward, the encapsulated starch was crushed and stored in a hot air oven for subsequent use.

Cell Cytotoxicity Assay and Morphological Identification

To investigate the effects on cytotoxicity, we used a concentration of 4 µM as a low concentration of CS and encapsulated NPs (CS, CMP, and CMA), and concentrations of 100 to 400 µM as high concentrations of CS. The effect of CS on cell viability was determined by the colorimetric MTT method. Briefly, 1. 5 × 10⁵ cells/mL of HSC-4 were cultured in a 96-well plate with varying (0, 100, 200, 300, 400, and 600 µM) concentrations of CS for 24 h. MTT solution at a concentration of 0. 5 mg/mL in 1x PBS was added to each well and incubated at 37 °C in the dark for 4 h. After incubation, the formazan crystals were dissolved in 100% DMSO and the absorbance of the formazan solution was measured at 570 nm using a microplate reader (BMG Labtech). The 50% inhibitory concentration (IC₅₀) was obtained from the dose–response curve of percent viability (Y) versus concentration tested (X). IC₅₀ was calculated by linear regression performed using Microsoft Excel. HSC-4 cells were placed in 6-well culture plates at a density of 1. 5 × 10⁵ cells/mL and their morphology was observed under an inverted microscope.

Western Blot Analysis

Protein expressions of Caspase-3, Cleaved Caspase-3, and receptor-interacting serine/threonine-protein kinase 3 (RIPK3) (all from Cell Signaling Technologies) were measured by Western blot analysis after the cells had been incubated with diluted CS and encapsulated NPs of CS. In brief, total proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer buffer. The concentration of proteins was evaluated by Bradford assay (Hercules). An equal amount of protein (30 µg) was subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. The proteins were subsequently transferred to a PVDF membrane (Millipore) and incubated with appropriate primary and secondary antibodies. The membranes were developed using x-ray film; the densitometry of the protein bands was quantified by ImageJ software and relatively normalized with β-actin (Cell Signaling Technologies).

RNA Isolation and Reverse Transcription ( RT )- PCR

After HSC-4 cells had been treated with 4, 100, 200, and 400 µM CS, and 4 µM nanoencapsulated CS (CS, CMP, and CMA) for 24 h, total RNA was isolated using the NucleoSpin RNA Plus kit (Macherey-Nagel), following the manufacturer's protocol, and 1 µg of RNA was used for complementary DNA (cDNA) synthesis using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo Co. , Ltd). PCR was performed in a BioRad/C1000Touch Thermocycle (BioRad) with specific primers. The amplified cDNA products were electrophoretically separated on a 1. 5% agarose gel and visualized by ethidium bromide (EtBr) staining. The relative expression level of a target gene was quantified by normalization with GAPDH gene as an internal control. Primers used in the experiment are shown in Table S1 (Supplementary Table1).

Detection of Supernatant and Cellular Protein Levels

To determine the supernatant and cellular protein levels and confirm necrotic cell death in HSC-4 cells, cultured cells were treated with 0, 4, 100, 200, and 400 µM CS for 24 h. In another experiment, the cells were treated with 4 µM of encapsulated NPs for 24 h. The supernatant was then collected, and cells were transferred into another microcentrifuge tube before centrifugation to wash the cell pellet. The treated cells were lysed using 10% DMSO at room temperature (RT) and vortexed to break the cells. The supernatant and cell lysates were assessed for total protein using the bicinchoninic acid (BCA) assay. ¹³ DMEM alone and containing 10% DMSO were used as the negative controls.

Measurement of Intracellular ROS Generation

To compare the effect of CS and encapsulated NPs in reactive oxygen species (ROS) generation on HSC-4 cells, cultured cells were treated with either various concentrations (0, 100, 200, and 400 µM) of CS or 4 µM of CS, CMP, and CMA, respectively for 24 h. The generation of ROS was evaluated using 10 µM of 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA), followed by incubation at 37 °C for 1 h in the dark. The ROS level was measured using fluorescence-associated oxidation of DCFH-DA to dichlorofluorescein (DCF). Samples were evaluated using a fluorescent microplate reader (Thermo Scientific Varioskan, USA) at excitation and emission wavelengths of 485 and 530 nm. Results are expressed as fluorescence relative to control.

MDC Assay

To analyze autophagy as a self-defense strategy in oral cancer cells, monodasylcadaverine (MDC) was used to label autophagic vacuole membrane lipids. Briefly, HSC-4 cells were cultured in 24-well plates at a density of 2 × 10⁵ cells/mL for 24 h. After treatment and washing, these cells were fixed with 4% PFA for 20 min at RT. Then, cells were washed with 1x PBS twice and incubated with 50 µM of MDC for 10 min at RT. Finally, cells were washed with 1x PBS, five times, before they were placed on a slide and observed under a fluorescent microscope. The fluorescence intensity and single-cell fluorescence counts were measured using automated counting in ImageJ.

Statistical Analysis

All data are expressed as mean ± SD. Different statistical comparisons were achieved by independent t-test by comparison between the two independent groups. Multiple comparisons among more than two experimental groups were analyzed by analysis of variance (ANOVA), followed by post hoc Duncan's test. The P-value of < . 05 was considered as a statistically significant difference.

Results and Discussion

The Effect of Cordycepin and Encapsulated Nanoparticles on Cell Proliferation and Morphology

Concentrations of CS ranging from 100 to 600 µM inhibited the proliferative activity of HSC-4 cells. The half-maximal inhibitory concentration (IC₅₀) of CS on HSC-4 was 360. 69 ± 11 µM (Figure 1A). Moreover, observation of 24 h-treated cells (Figure 1B) with a high dose of CS (100-400 µM) revealed cells with an irregular cellular morphology including cellular swelling and membrane rupture, and increased occurrence of cell death in a dose-dependent manner. It was also found that the loss of cell membrane integrity and cell death led to the uncontrolled release of cell products into the extracellular space. On the other hand, cell death with a regular cycle morphology indicating apoptosis was observed in cells after being treated with encapsulated NPs for 24 h, while a low dose of CS (4 µM) did not lead to cell death (Figure 1C). These results demonstrate that necrotic cell death was induced by a high dose of CS, but not with a low dose. Interestingly, encapsulated NPs did not induce necroptosis, but rather induced apoptosis.

Figure 1.

(A) Cell viability of human oral cancer cells (HSC-4) posttreatment with cordycepin (CS). (B) and (C) The morphology of HSC-4 cells posttreatment with a high concentration (100-400 µM) of CS or low concentration (4 µM) of either CS or encapsulated nanoparticles (NPs). The arrows show cell swelling and membrane rupture, exhibiting necroptotic cell death. Data are presented as mean ± SD (n = 3). ***P < . 001 versus control cells.

Some related studies include one on quercetin that induces necrosis and apoptosis in human oral squamous carcinoma SCC-9 cells, ¹⁴ and one on the water-soluble fraction of frankincense that is able to induce necrosis and apoptosis in an OSCC cell line (KB cells). ¹⁵ These findings confirm that necrosis can occur in cancer cells. Necrosis of HSC-4 cells, however, could have a role in cancer progression since it releases cellular contents that may exert tumor-promoting activity. ¹⁶ The findings that CS NPs switched HSC-4 cell death from necroptosis to apoptosis suggest that nanoencapsulation of CS provides improved biological activity for HSC-4 cell death induction and may be more suitable for therapeutic use.

Expression of Apoptotic, Autophagy, Necroptotic, and Pyroptotic Genes in HSC - 4 Cells After Treatment

The expression levels of specific genes were determined to identify alterations in the cancer signaling pathways of HSC-4 cells treated with CS and encapsulated NPs. A high dose of CS caused significant decrease in apoptosis-related genes (BAX, Caspase-3, Caspase-8, and Caspase-9) (Figure 2A and C), and decrease in autophagy and pyroptotic genes, LC3, ATG12, Caspase-1 (Figure 3A and C). Conversely, a low dose of CS led to a significant increase in apoptotic gene expression (Figure 2B and D) including BAX, Caspase-8, and Caspase-3, and a slight increase in autophagy genes such as LC3 (Figure 3B and D). These results demonstrate that although a low dose of CS is able to stimulate apoptotic genes, it is not enough to cause apoptosis, as there was no significant difference in cell proliferation when compared to the control. On the other hand, encapsulated NPs led to a significant increase in P53, BAX, Caspase-3, and Caspase-8 (Figure 2B and D) and a significant decrease in RIPK3, P62, ATG12, and Caspase-1 (Figure 3B-D). These results suggest that encapsulated NPs can cause cellular apoptosis through the activation of caspases and the inhibition of autophagy activation.

Figure 2.

Expression of apoptotic genes (P53, BAX, Caspase-3, Caspase-8, and Caspase-9) of human oral cancer (HSC-4) cells posttreatment. (A) and (C) mRNA expression levels of genes relative to controls (fold) after HSC-4 cells were treated with either a high concentration (100-400 µM) or (B) and (D) a low concentration (4 µM) of cordycepin (CS) or encapsulated nanoparticles (NPs). (E) and (F) The ratio of Cleaved Caspase-3/Caspase-3 protein expression levels after HSC-4 cells were treated with a high concentration (100-400 µM) of CS and low concentration (4 µM) of either CS or encapsulated NPs, respectively. Values are expressed as mean ± SD (n = 3). Significance versus control cells is indicated as follows: *P < . 05, **P < . 01.

Figure 3.

Expression of autophagy (P62, LC3, Atg5, and Atg12), necrotic (RIPK3), and pyroptotic (Caspase-1) genes of human oral cancer (HSC-4) cells posttreatment. (A) and (C) mRNA expression levels of genes relative to controls (fold) after HSC-4 cells were treated with high (100-400 µM) and (B) and (D) low concentrations (4 µM) of either cordycepin (CS) or encapsulated nanoparticles (NPs). (E) and (F) Expression levels of RIPK 3 protein after HSC-4 cells were treated with either a high (100-400 µM) or low concentration (4 µM) of either CS or encapsulated NPs, respectively. Values are expressed as mean ± SD (n = 3). Significance versus control cells is indicated as follows: *P < . 05, **P < . 01, ***P < . 001.

We next determined the levels of protein expression by Western blotting. It was noticed that the expression of Cleaved Caspase-3/Caspase-3 ratio was significantly increased after being treated with high doses (100-400 µM) of CS and encapsulated NPs, while a low concentration of CS did not affect this ratio (Figure 2E-F). On the other hand, high concentrations of CS and encapsulated NPs significantly caused a decreased RIPK 3 expression whereas a low dose of CS did not produce any effect on the expression level of RIPK 3 protein (Figure 3E-F).

Programmed cell death (PCD) can be mainly classified into apoptosis, autophagy, necroptosis, and pyroptosis. PCD is different from necrosis and is tightly regulated by a cascade of gene expression events. ¹⁷ The expression of apoptotic, autophagy, necroptotic, and pyroptotic genes in HSC-4 cells corresponded to cell death pathways. High concentrations of CS-induced necrosis of HSC-4 cells by inhibiting the expression of apoptotic genes (BAX, Caspase-3, Caspase-8, and Caspase-9) whereas three forms of nanoencapsulation of CS (CS, CMP, and CMA) increased the expression of apoptotic genes, including P53, BAX, Caspase-3, and Caspase-8 in HSC-4 cells. Encapsulated CS also inhibited the expression of the anti-apoptotic gene BCL2.

Caspases and RIPK are critical regulators of apoptosis and necroptosis. ¹⁸ Specifically, Caspases 3, 8, and 9 have a well-established role in mediating apoptosis. However, necroptosis is a caspase-independent death pathway. It requires the kinase activity of RIPK1 and RIPK3. It has also been suggested that the activity of Caspase-8 represents a central switch regulating cell death plasticity between apoptosis and necroptosis. ¹⁹ The findings that nanoencapsulation of CS increased the expression of apoptotic genes, especially Caspase-8, and inhibited the expression of the necrotic gene RIPK3 suggest that nanotechnology could enhance the bioactivity of CS by switching the cell death pathway from necroptosis toward apoptosis.

Cordycepin Dose Affects the Protein Content in the Supernatant and Cellular Fraction

To confirm the presence of necrotic and apoptotic cell death, HSC-4 cells were treated with either CS or encapsulated NPs. Then, the supernatant and cellular protein levels were determined using BCA assay, since the plasma membrane of necrotic cells is commonly destroyed early, whereas that of apoptotic cells is near-to-contact until a late stage. HSC-4 cells treated with CS (Figure 4A left) had a significantly increased supernatant protein level, while the cellular protein level was significantly decreased, indicating necrosis. In contrast, in cells treated with encapsulated NPs (Figure 4A right), a significantly decreased protein level in the supernatant and a slightly higher level in the cellular compartment were observed, indicating apoptosis.

Figure 4.

(A) The level of supernatant and cellular proteins on human oral cancer cells (HSC-4) posttreatment with either a high (100-400 µM) or low concentration (4 µM) of either cordycepin (CS) or encapsulated nanoparticles (NPs). (B) The level of ROS generation in HSC-4 cells posttreatment. (C) mRNA expression levels of GPX, SOD, and CAT genes relative to controls (fold) after treatment of HSC-4 cells. (D) Accumulation of monodasylcadaverine (MDC)-labeled vacuoles on HSC-4 cells after labeling. The relative fluorescence intensity of MDC and the level of single fluorescence count of MDC on HSC-4 posttreatment were determined by ImageJ and MDC staining was observed using a fluorescent microscope. Values are expressed as mean ± SD (n = 3). Significance versus control cells is indicated as follows: *P < .05, **P < .01 and ***P < .001.

Apoptosis plays an important role in physiological conditions, while necrosis is considered highly inflammatory and stressful. It is conceivable that apoptosis and necrosis can be activated by herbal medicine and may have therapeutic potential in a variety of diseases, including cancer treatment. ²⁰ The detection of protein inside and outside of HSC-4 cells was performed. In cells treated with a high concentration of CS, increased supernatant and decreased cellular proteins were observed, indicating membrane rupture after necrosis. On the contrary, those treated with encapsulated NPs exhibited decreased secretion and slightly increased intracellular proteins when compared to the control. It may be possible that protein synthesis in the cell was reduced because the high-dose CS-treated cells were in a stressful state. ²¹ In addition, an effect of encapsulated CS NPs on protein secretion and intracellular protein levels may be due to the modulation of autophagy, causing less secretion of proteins. ²² Modulating autophagy by encapsulated NPs might reduce autophagy-degraded intracellular proteins, while activation of autophagy by CS treatment might degrade intracellular proteins and subsequently release them into the culture media.

Comparison of the Effectiveness of a High Dose of Cordycepin and Encapsulated Nanoparticles in ROS Generation

To compare the effectiveness of a high dose of CS and encapsulated NPs on the stimulation of ROS generation, HSC-4 cells were treated with various concentrations (0, 100, 200, and 400 µM) of either CS or 4 µM of CS, CMP, and CMA, respectively for 24 h. Significantly increased relative florescence intensity was demonstrated after CS treatment compared with control. Encapsulated NPs containing a small amount of CS increased ROS generation in HSC-4 cells, more than CS before modification (Figure 4B). However, a high dose of CS (Figure 4C) significantly decreased antioxidant genes (SOD, GPX, and Catalase). These results demonstrate that ROS involves both apoptotic and necrotic cell death induced by encapsulated NPs of CS and CS before modification. However, apoptotic cell death might require a higher level of ROS than necrotic cell death.

This can be explained because a high dose of CS-induced necrosis in HSC-4 cells, as they were unable to stimulate antioxidant gene expression because of their damaged DNA from oxidative stress, ²³ whereas encapsulated NPs stimulated antioxidant genes ¹¹ and thereby prevented cells from undergoing necrosis.

Cordycepin Stimulates Autophagy as a Self - Defense Strategy in HSC - 4 Cells

Since a low concentration of CS increased the expression of the LC3 gene, it may be possible that oral cancer cells stimulate autophagy modulation, resulting in their survival from a low concentration of CS. To elucidate the mechanism of self-defense in HSC-4 cells through the expression of autophagy, HSC-4 cells were treated with either CS or encapsulated NPs and then assessed for MDC staining by fluorescence spectroscopy (Figure 4D). These results showed that the intensity of MDC staining and the number of single fluorescent cells after CS treatment significantly increased in a dose-dependent manner, while encapsulated NPs significantly decreased MDC staining when compared to the control. These results demonstrate that CS-induced autophagy for HSC-4 cell survival, such that a low concentration of CS was unable to affect the proliferation of HSC-4 cells. Moreover, it was found that a high dose of CS led to a higher amount of fluorescence, which was related to the increased expression of necroptotic genes. These results show that the autophagy membrane is also associated with necrotic cell death via autophagy modulation. On the contrary, we found only a small amount of MDC fluorescence in HSC-4 cells treated with encapsulated NPs when compared to the control. These results demonstrate that encapsulated NPs inhibited the process of autophagy and necrosome development in HSC-4 cells.

In the low-dose stimulation model, CS induced a slight expression of autophagy genes (P62, LC3, and ATG5). Previous studies have shown that autophagy is involved in the promotion of tumorigenesis and the development of cancer, and alterations in autophagic signaling pathways are frequently found in cancer. ²⁴ Therefore, although HSC-4 cells treated with a low concentration of CS exhibit increased level of apoptotic genes, the self-defense mechanism of these cells is associated with autophagy modulation. ²⁵ The MDC assay affirmed that autophagy modulation occurred after CS treatment, but not with encapsulated NPs. This suggests that HSC-4 cells could survive a low dose of CS through autophagy modulation. With a high dose of CS, the cells were necrotic because oxidative stress damaged their DNA components and proteins, leading to a high level of MDC fluorescent label in the membrane. It has been noted that autophagy genes, including p62, LC3, and ATG5, may play a role in the formation of the necrosome on autophagosomes in low-dose CS-treated HSC-4 cells. Taken together, the autophagy process was inhibited when cells were treated with encapsulated NPs, which inhibited the growth of HSC-4 cancer cells.

Conclusion

This study provides a deeper understanding of the signaling pathways active in oral cancer cells and the strategy by which they protect themselves from the anti-cancer activity of CS. Moreover, the mechanisms by which encapsulated NPs inhibit the growth of HSC-4 cells were elucidated. We also confirmed the inhibitory mechanisms of CS-loaded CSNPs on cancer cells through the activation of apoptotic cell death; CS alone causes necrotic cell death at a high dose, but at a low dose does not result in cell death (Figure 5). Our results show that nanotechnology and encapsulation may be able to develop natural extracts or other biologically active substances in cassava starch to be used as drug delivery systems for both normal and cancer cells. In addition, it is also expected that NPs will be used to enhance medical treatments in the future.

Figure 5.

Schematic diagram representing the different signaling pathways active and the strategy by which they protect themselves from the anti-cancer activity of low dose, high dose, and encapsulated nanoparticle (NP) of cordycepin (CS) in oral cancer cells.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221074838 - Supplemental material for Nanoencapsulation of Cordycepin Induces Switching from Necroptosis to Apoptosis in Human Oral Cancer Cells (HSC-4) Through Inhibition of Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3) and Autophagy Modulation

Supplemental material, sj-docx-1-npx-10.1177_1934578X221074838 for Nanoencapsulation of Cordycepin Induces Switching from Necroptosis to Apoptosis in Human Oral Cancer Cells (HSC-4) Through Inhibition of Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3) and Autophagy Modulation by Palakorn Kaokaen, Nipha Chaicharoenaudomrung, Phongsakorn Kunhorm, Kedkanya Mesil, Thunwa Binlateh, Parinya Noisa and Paiboon Jitprasertwong in Natural Product Communications