Biomedicines | Free Full-Text | Shrimp Lipids Inhibit Migration, Epithelial–Mesenchymal Transition, and Cancer Stem Cells via Akt/mTOR/c-Myc Pathway Suppression
1. Introduction
Lung cancer has long been recognized as a malignant tumor with a high mortality rate, among which non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases [1,2]. Although surgery combined with radiotherapy, chemotherapy, and targeted therapy has achieved certain results in treating NSCLC, the survival rate of advanced patients is relatively low [3,4]. The metastasis of lung cancer is one of the main causes of treatment failure and patient mortality [5]. Therefore, understanding the mechanism of cancer cell metastasis is extremely important.
The metastasis in many cancers is linked with the cellular process of epithelial–mesenchymal transition (EMT). This process involves the transformation of cells into mesenchymal motile cells and the loss of cell adhesion of the cells, which trigger a variety of pathogenic characteristics [5]. Several proteins are involved in the EMT process and contribute to cancer progression and metastasis, such as Snail, Slug, and Vimentin [6]. Transcription factors such as Snail and Slug are known to regulate the expression of EMT genes and play a crucial role in triggering the EMT process [6]. Overexpression of these transcription factors has been shown to be associated with poor prognosis and metastasis in many cancers, including lung cancer [7]. Vimentin is a mesenchymal marker that is upregulated during EMT and is associated with increased motility and invasiveness of cancer cells [5]. Recent studies have found that EMT is one factor that can induce cancer stem cell (CSC) characteristics. CSCs are among the most important factors contributing to cancer recurrence, metastasis, and drug resistance [8,9].
Many studies indicate that lung CSCs maintain their stemness, or stem cell-like properties, through their sustained expression of specific cell surface markers such as glycoproteins prominin-1 (CD133) and CD44-antigen (CD44), transcription factors like Sox2, and several signaling pathways [9,10]. These biomarkers are also associated with EMT and are involved in regulating the initiation and development of tumors [11]. The function of Sox2 involves regulating stem cell self-renewal and differentiation [12]. Furthermore, upregulation of Sox2 in a hypoxic environment can promote CD133 expression in lung cancer cells [13].
The upstream mechanism and the major controlling signaling pathway that determine stem cell properties in cancer have been extensively studied, especially as CSCs have emerged as key targets for novel anticancer therapeutics [11,14]. ATP-dependent tyrosine kinase, or protein kinase B (Akt), has been shown to regulate CSC properties in cancer [14]. The activation or augmentation of the active Akt signal has been shown to induce CSC phenotypes and promote the survival of the CSC population [15]. In addition, the stem cell transcription factors, including Oct4, Sox2, and Nanog, have been demonstrated to be downstream targets of the Akt signaling pathway [16]. The activation of the Akt signaling pathway promotes the formation of tumor-initiating cells by inducing EMT and facilitating cancer cell migration and invasion [17]. The suppression of the mammalian target of rapamycin (mTOR), the downstream target of Akt signaling, has been found to reduce invasion and cell migration as well as block CSCs from forming in NSCLC [18,19]. Moreover, c-Myc is a downstream target of the Akt/mTOR pathway, and its expression has been linked to both the EMT process and stem cell-like properties in cancer [20].
Shrimp byproducts, including the heads, shells, tails, and other parts of the shrimp that are not typically consumed as food, can make up to 50% of the shrimp’s total weight, and are often discarded as waste, raising environmental concerns [21]. A shrimp head contains oil or lipids, which are primarily composed of astaxanthin and various types of fatty acids, including polyunsaturated fatty acids (PUFAs) as well as cholesterol [22]. Astaxanthin has been widely investigated for its potential pharmacological properties such as antioxidant, antimicrobial, anticancer, anti-inflammatory, cardiovascular, antidiabetic, and skin-protective effects [23,24]. Essential fatty acids, including omega-3 PUFAs, are crucial for human health as they can help reduce the risk of heart disease and decrease inflammation [25]. Studies have reported that omega-3, particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can inhibit cancer cell growth, induce cancer cell apoptosis, and exert an anti-angiogenic effect in several types of cancers [26,27,28]. Direct treatment with n-3 PUFAs can inhibit the growth of breast cancer cells and enhance differentiation [29]. The pro-differentiating effect of DHA was also confirmed in the human melanoma cell model [30]. However, epidemiological studies revealed that excessive cholesterol consumption raised the risk of cardiovascular disease and stroke [31,32] and cancer by encouraging cell proliferation, invasion, and metastasis in gastric, breast, and lung cancers [33,34,35]. Therefore, eliminating cholesterol can improve the quality of shrimp lipids. The purpose of this study was to investigate the effect of cholesterol-free shrimp lipids (CLs) on several key aspects of cancer progression, including cancer cell migration, EMT, and the expression of CSC-like phenotypes in NSCLC cell lines H460 and H292, as well as to identify the molecular mechanisms underlying these effects.
2. Materials and Methods
2.1. Preparation of Cholesterol-Free Shrimp Lipids (CLs)
2.1.1. Shrimp Lipid Extraction and Fractionation Using Ethanol
The shrimp species used in this study was Litopenaeus vannamei, and it was obtained from Sea Wealth Frozen Food Co., Ltd. (Songkhla, Thailand), under frozen conditions (−18 °C). After thawing the shrimp heads, they were ground in a blender (National, Tokyo, Japan) to create a uniform paste. The method developed by Ei and coworkers [36] involved using a hexane/isopropanol mixture (1:1) to extract lipids from the shrimp paste. After extracting the lipids, the shrimp lipid extract was fractionated using ethanol in a separating funnel. The fractionation process involved adding ethanol to the lipid extract twice and separating the polar and non-polar lipid fractions. The lower layer of the non-polar lipid fraction was dissolved in 5 mL of hexane and kept at −20 °C, while the upper layer of the polar lipid fraction was evaporated using a rotary evaporator [37].
2.1.2. Preparation of Silica Column
A glass column (diameter: 2 cm; height: 35 cm) was packed with dried silica gel (pore size: 7–230 mesh). Before packing, the bottom of the column was sealed using a pinch of cotton, which was then covered with a layer of celite (1 g) dispersed in 50 mL of hexane. Afterward, dried silica gel mixed with hexane was poured into the column and allowed to settle. Then, the column was purged with nitrogen gas to remove any air bubbles. Finally, the top of the silica gel was covered again with 1 g of celite. To prevent dryness and moisture absorption, the column was filled with hexane until it was used [37].
2.1.3. Fractionation of Polar Lipids
Different fractions of shrimp lipids were separated through the silica gel column using different solvents at several ratios [22]. Oil was first extracted from a shrimp cephalothorax [38]. Then, polar lipids were separated from the shrimp oil via ethanol crystallization at −20 °C. Thereafter, the obtained polar lipids (350 mg) were dissolved in the minimum volume of hexane, allowing them to settle at the top of the celite layer and then pass through the silica gel using hexane (100 mL), in which the first fraction (F1) was collected. To acquire the second, third, fourth, and fifth fractions, namely F2, F3, F4, and F5, respectively, the hexane/acetone mixture at varying ratios of 98:2, 96:4, 94:6, and 92:8 (v/v), respectively, was passed through the column. During loading, solvents were poured slowly around the wall of the column without disturbing the gel. Elution was accomplished when the reddish orange color in the eluent disappeared. The final fraction (F6) was eluted using methanol. The elution was performed at a constant flow rate of 4.5 mL/min. All the fractions were collected, and the elution volume for each fraction was recorded. Subsequently, evaporation was carried out using a rotary evaporator (Tokyo Rikakikai, Tokyo, Japan). The CLs were obtained by mixing non-polar lipids and fractions F1, F4, F5, and F6 (Figure 1). The mixed lipids were flushed with nitrogen, stored in an amber bottle, and tightly capped before being placed at −40 °C.
2.2. Fatty Acid Composition of CLs
The fatty acid composition of the sample was assessed using the Raju and Benjakul [22] technique. Initially, 10 mg of CLs was dissolved in 1 mL of hexane, followed by esterification through the addition of 200 µL of a 2 M methanolic sodium hydroxide solution at 50 °C for five min. After cooling, 200 µL of a 2 M methanolic hydrochloric acid solution was added. This mixture underwent 10 min of centrifugation at 3500× g, yielding a hexane phase for analysis. The hexane phase was introduced into an Agilent GC 7890B gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) with an injection temperature of 250 °C. The chromatographic conditions included an initial column temperature of 80 °C, with a programmed 4 °C/min increase over 40 min to 220 °C, and then to 240 °C. The compounds were detected at a flame ionization detector temperature of 270 °C, and the peaks were identified using authentic standards. The obtained results were expressed in terms of grams per 100 g (g/100 g). Table 1 provides a comprehensive presentation of the fatty acid composition and content of the CLs extracted from the cephalothorax of Pacific white shrimp, including their respective quantitative values.
2.3. Preparation of CL Stock Solution
CL 100 mg/mL stock solutions were prepared by dissolving the CLs in dimethyl sulfoxide (DMSO) and storing them at −20 °C. To achieve the experimental concentrations (ranging from 0 to 500 μg/mL), the stock solution was diluted in a culture medium. DMSO content in the final solution was 0.2%.
2.4. Cell Cultures
Human NSCLC H460 and H292 cells and human keratinocyte HaCaT cells were incubated at 37 °C with 5% CO2, which provided a controlled environment for cell growth and survival. H460 and H292 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, USA), whereas HaCaT cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA). The RPMI and DMEM were supplemented with 2 mM of L-glutamine (Gibco, Grand Island, NY, USA) and 10% fetal bovine serum (FBS) (Merck KGaA, Darmstadt, Germany).
2.5. Cell Viability Assay
H460, H292, and HaCaT cells were seeded in 96-well plates at 1 × 104 cells per well and allowed to adhere for 16 h at 37 °C. Cells were treated with various concentrations of CLs (0–500 µg/mL) at varying time periods of 24, 48, and 72 h. A total of 100 µL of MTT solution (0.4 mg/mL) was added to each well. The plates were then incubated for 3 h at 37 °C. The MTT solution was removed, and the formazan crystals were dissolved in 100 µL of DMSO. The intensity of the purple formazan was then assessed using a microplate reader at a wavelength of 570 nm.
2.6. Nuclear Staining Assay
For screening apoptotic cell death using double staining with Hoechst 33342 and propidium iodide (PI), H460, H292, and HaCaT cells (1 × 104 cells/well) were seeded onto 96-well plates for 12–16 h. The cells were treated with various doses of CLs (0–500 µg/mL) for 24, 48, and 72 h. The cells were then labeled with Hoechst 33342 (10 g/mL) and PI (5 g/mL) for 30 min at 37 °C. The staining allowed for the visualization and imaging of apoptotic cells, which appeared bright blue due to the Hoechst 33342 staining, while necrotic cells appeared red due to the PI staining using fluorescence microscopy (Nikon ECLIPSE Ts2; Tokyo, Japan).
2.7. Wound-Healing Assay
NSCLC cells (3 × 104 cells/well) were seeded onto 96-well plates for 24 h at 37 °C. A wound was produced by creating a gap in a straight line on the cell monolayer in each well using a 20 μL sterile plastic micropipette tip. The cells were then treated with various doses of CLs (0–500 µg/mL) for a period of up to 72 h. Images of the cells were taken at the specified time points of 0, 24, 48, and 72 h after treatment, and the wound space was measured using Image J software version 1.52a.
2.8. Migration Assay
A transwell migration assay was performed to test the migratory activity of the cells. The cells were treated for 48 h. Then, the cells were trypsinized and resuspended in serum-free media. The lower chamber of each transwell insert was filled with 600 µL of RPMI media containing 10% FBS, and the upper chamber of the insert was then filled with cells at a density of 1 × 105 cells per well. After 16 h, the culture medium was removed from the transwell inserts, and the cells were washed twice with PBS to remove any residual media and serum. The cells were then fixed with 3.7% formaldehyde for 15 min to preserve their cellular structure and prevent any further migration. The cells were then permeabilized with 100% methanol for 15 min to allow for crystal violet staining to penetrate the cells and adhere to the cellular components. Next, the cells were stained with 0.5% crystal violet in 25% methanol for 10 min. The membranes were washed several times with PBS to remove any excess dye. The cells that remained in the upper chamber of the transwell insert were removed using cotton swabs. Finally, images of the cells that moved through the insert’s pores were captured using a 10× light inverted microscope.
2.9. Western Blot Analysis
Western blot analysis was used to detect and quantify specific proteins in a sample. NSCLC cells (H460 and H292) were plated at a density of 2 × 105 cells per well in 6-well plates at 37 °C for 24 h and treated with various doses of CLs (0–500 µg/mL) for 48 h. The cells were washed with cold PBS and lysed using RIPA buffer containing protease inhibitors, Triton X, and PMSF for 40 min on ice. The total protein content of the cell lysate was quantified using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of protein sample were loaded onto SDS polyacrylamide gel and electrophoresed. The proteins were then transferred from the gel onto a polyvinylidene difluoride (PVDF) membrane using an electric field. The membrane was then blocked with 5% skim milk in TBST for 1 h to prevent non-specific binding of antibodies to the membrane. Next, primary antibodies, Vimentin (Cell Signaling, #5741, 1:1000), Snail (Cell Signaling, #3879, 1:1000), Slug (Cell Signaling, #9585, 1:1000), mTOR (Cell Signaling, #2983, 1:1000), p-mTOR ser2448 (Cell Signaling, #5536, 1:1000), Akt (Cell Signaling, #9272, 1:1000), p-Akt ser473 (Cell Signaling, #4060, 1:1000), CD44 (Cell Signaling, #3570, 1:1000), c-Myc (Cell Signaling, #18583, 1:1000), CD133 (Abcam, #ab19898, 1:1000), Sox2 (Cell Signaling, #3579, 1:1000), and β-actin (Cell Signaling, #4970, 1:1000), were incubated with the membrane overnight at 4 °C. The primary antibody bonded to the protein, and any unbound antibody was washed three times with TBST. A secondary antibody conjugated to an enzyme, or a fluorescent tag, was then added and incubated for 2 h at ambient temperature. Finally, protein bands were detected by adding a chemiluminescent substrate and were exposed to X-ray film. The intensity of the protein bands was quantified using Image J software to determine the level of protein expression in the sample.
2.10. Immunofluorescence Assay
Cells were seeded in a 96-well plate at a density of 5 × 103 cells/well, and they were incubated overnight to allow them to adhere to the plate. After the cells were treated with CLs (0–500 µg/mL) for 48 h, they were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. Next, they were permeabilized with 0.5% Triton-X in PBS for 5 min and blocked with 10% FBS in PBS for 1 h. They were then probed with primary antibodies (Vimentin, Snail, Slug, mTOR, p-mTOR, Akt, p-Akt, CD44, c-Myc, CD133, and Sox2) overnight at 4 °C. After incubation with the secondary antibody and staining with Hoechst 33342 for 1 h at room temperature, the cells were rinsed with PBS, fixed with 4% paraformaldehyde in PBS, and mounted using 50% glycerol. The images were obtained using a fluorescence microscope, and Image J software was used to analyze the fluorescence intensity of stained cells.
2.11. Spheroid Formation Assay
Cells were pre-treated with CLs (0–500 µg/mL) for 48 h, and then they were plated at a density of 5 × 103 cells per well in a 24-well ultra-low attachment plate. Primary tumor spheroids were captured after incubation for 3 and 7 days by a phase-contrast microscope (Nikon ECLIPSE Ts2; Tokyo, Japan). Then, spheroids were trypsinized and resuspended into a single cell. The 5 × 103 cells/mL were seeded onto 24-well ultra-low attachment plates for 10 days to form secondary CSC-enriched spheroids and photographed at day 3, day 7, and day 10. The secondary spheroids were then transferred to a 96-well ultra-low attachment plate, with one spheroid per well, and treated with various concentrations of CLs. The changes in their size and shape were monitored at 0, 1, and 2 days using an inverted microscope. The single spheroids were stained with Hoechst 33342 and PI for 15 min on day 2, to assess their viability and proliferation, and imaged using a fluorescent microscope.
2.12. Statistical Analysis
The mean ± standard deviation (SD) of three independent results was reported. GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) was used to conduct an analysis of the statistical differences between the groups using ANOVA, followed by Dunnett’s multiple comparisons test for individual comparisons. A graph of all data was also generated. A p-value of less than 0.05 was considered statistically significant.
4. Discussion
Shrimp heads, well known as a byproduct of the shrimp processing industry, contain high levels of carotenoids, including astaxanthin and astaxanthin esters. These compounds are powerful antioxidants that have been linked to several health benefits, such as reducing inflammation, improving immune function, and suppressing the development of different types of cancer [22,23,24]. Additionally, shrimp head oil is rich in PUFAs, particularly EPA and DHA, which are essential omega-3 fatty acids crucial for maintaining heart health and brain function and reducing inflammation [25]. PUFA uptake has also been inversely related to lung, prostate, breast, and colorectal cancers [44,45]. In lung cancer, several studies have shown the potential effects of omega-3 and omega-6 PUFAs in suppressing proliferation, promoting apoptosis, and inhibiting angiogenesis [46,47]. Moreover, n-3 PUFAs enhanced the sensitivity of breast cancer, sarcoma, and leukemia in in vivo models of chemotherapeutic agents [28]. However, the consumption of shrimp oil and lipids has been restricted due to the presence of cholesterol. The American Heart Association recommends that individuals consume no more than 300 mg of cholesterol per day [48]. Nevertheless, individuals with high blood cholesterol levels, diabetes, or a history of cardiovascular disease may need to further reduce their intake to no more than 200 mg/day.
Migration and invasion of cancer cells are major factors that contribute to the poor prognosis of many types of cancer [49]. Targeting the process of EMT, which is associated with the migration and invasion of cancer cells, is an important strategy for developing new cancer therapies to treat metastasis. In lung cancer, EMT plays a critical role in cancer progression and metastasis [7]. By inhibiting this process, it may be possible to prevent cancer cells from acquiring the migratory and invasive properties needed for metastasis. There are several potential targets for inhibiting EMT in lung cancer, including various signaling pathways and transcription factors that regulate EMT-related gene expression [8,9]. For example, the TGF beta signaling pathway has been implicated in EMT induction and cancer metastasis [50]. Inhibitors targeting this pathway are currently under development as potential cancer therapeutics. Additionally, several transcription factors, such as Snail, Slug, and Twist, are known to regulate EMT gene expression and are potential targets for cancer therapy [8]. In this study, CL treatment inhibited the migratory activity of human NSCLC (Figure 3A–C). From a mechanistic approach, CLs were shown to inhibit EMT in these cells, as indicated by the depletion of Vimentin, a hallmark of mesenchymal cells, and the deregulation of the levels of the EMT transcription factors Snail and Slug in both H460 and H292 cells (Figure 4A–D). These results are consistent with previous studies showing that astaxanthin and PUFAs, which are dominant compounds derived from shrimp lipids, suppress cancer cell migration [51,52].
CD133 and CD44 are cell surface markers commonly used to identify CSCs [9,10]. CD133, also known as Prominin-1, is a transmembrane glycoprotein that plays a crucial role in tumor recurrence, metastasis, and resistance to chemotherapy and radiation therapy [53]. CD44, on the other hand, is a transmembrane glycoprotein that is involved in cell–cell interactions, cell adhesion, and cell migration [54]. These biomarkers have been shown to be overexpressed on the surfaces of CSCs in several types of cancer, including breast, pancreatic, and lung cancers [53,54]. Studies have shown that the upregulation of CD133 and CD44 in lung cancer cells is associated with the induction of EMT, which is a critical step in the development of metastasis [9,10,11]. The suppression of CD133 and CD44 expression can decrease the formation of tumors and spheres, which are indicative of the self-renewal capacity of CSCs. In lung cancer cells, the downregulation of CD133 and CD44 expression has been shown to reduce the number of tumor spheres formed in vitro and to decrease tumor growth in vivo [10,55]. Sox2 is a transcription factor that plays a critical role in the self-renewal and maintenance of stem cells, including CSCs [12]. Sox2 has also been identified as a key regulator of the stemness and tumorigenic potential of CSCs in several forms of tumors [56]. Inhibition of Sox2 has demonstrated promising results in targeting lung cancer CSCs [57]. A recent report by Chen et al. showed that the knockdown of Sox2 expression using small interfering RNA (siRNA) in lung cancer CSCs led to a reduction in tumor sphere formation and cell proliferation [58]. Additionally, it increased sensitivity to cisplatin, a commonly used chemotherapy drug. These findings suggest that targeting CD133, CD44, and Sox2 expression or downstream signaling pathways might be promising strategies for developing new cancer therapies that target lung cancer CSCs. In the current study, we found that the expression of stemness markers CD133, CD44, and Sox2 was downregulated in response to CL treatment in both H460 and H292 cells (Figure 5A–D). Additionally, the CL treatment suppressed spheroid formation and induced lung CSC death by decreasing the expression of CD44 and CD133 (Figure 8 and Figure 9). To support the effect of CLs in inhibiting CSC, it was reported that astaxanthin has the potential to reduce the populations of BT20 and T47D breast CSCs and suppress stemness markers [59]. Similarly, DHA and EPA were found to suppress the development of cancer stem-like cells in colorectal cancer by inducing apoptosis [60].
Activated Akt is essential for the EMT process, which is a critical step in cancer metastasis [61]. Akt has been shown to regulate EMT by controlling the expression of various EMT-related transcription factors, such as Snail, Slug, and Twist [17]. In addition, Akt signaling has been shown to regulate CSC self-renewal and maintenance in various types of cancers, including lung cancer [14]. Akt inhibition has been shown to decrease the expression of stem cell markers such as CD133, CD44, and ALDH and to reduce the proportion of CSCs in lung cancer cell lines and patient-derived xenografts [10,62]. Studies have shown that inhibiting Akt signaling can lead to the downregulation of Sox2 expression and the depletion of lung CSCs in NSCLC [16]. Similarly, the suppression of mTOR signaling has been found to reverse EMT and decrease the CSC phenotype in lung cancer cells, as mTOR is a downstream target of Akt [19]. The Akt/mTOR signaling pathway can activate c-Myc, which plays a critical role in promoting the self-renewal and survival of CSCs in various cancers, including lung cancer [63]. c-Myc has been shown to regulate the expression of pluripotent transcription factors, such as Oct4, Sox2, and Nanog, and to promote the proliferation and survival of CSCs [16]. In addition, c-Myc can induce angiogenesis, promoting tumor growth and metastasis. In agreement with these observations, this study showed that CLs decreased the amounts of active Akt, mTOR, and c-Myc (Figure 6 and Figure 7), subsequently reducing EMT markers Vimentin, Snail, and Slug (Figure 4) and inhibiting CSC phenotype acquisition (Figure 8 and Figure 9). This suggests that the Akt/mTOR/c-Myc pathway and its downstream targets, including EMT and CSC markers, were regulated by the CLs. In addition, this comparison revealed that CL treatment produced comparable outcomes to earlier research on cancer development (Table 2). These effects parallel the findings observed with astaxanthin and PUFAs, suggesting potential similarities in their mechanisms of action.
Consistent with our observations, numerous other studies have investigated the effects of PI3K/Akt and mTOR inhibitors on cancer progression [42,64], further supporting the significance of Akt and mTOR inhibition in mediating the observed effects of CLs. LY294002, a specific inhibitor of the PI3K/Akt pathway, has been extensively studied in various cancer models, where it has demonstrated its efficacy in suppressing Akt signaling and downstream oncogenic processes [64]. LY294002 has demonstrated its efficacy in inhibiting cell proliferation, inducing apoptosis, and suppressing tumor growth, while also attenuating EMT by suppressing the Akt-mediated regulation of EMT-related transcription factors and signaling pathways [65]. Similarly, rapamycin has exhibited potent anticancer effects through the inhibition of mTOR signaling, leading to reduced tumor growth and metastasis [42,43]. In addition, both LY294002 and rapamycin have been implicated in modulating c-Myc expression in various cancer types [64,66].
