molecules

Article Substrate 1 Is Involved in the Phycocyanin-Mediated Antineoplastic Function of Non-Small Cell Cells

Shuai Hao, Qiancheng Li, Yuanpu Liu, Fannian Li, Qi Yang, Jing Wang and Chengtao Wang *

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing 100048, China; [email protected] (S.H.); [email protected] (Q.L.); [email protected] (Y.L.); [email protected] (F.L.); [email protected] (Q.Y.); [email protected] (J.W.) * Correspondence: [email protected]

Abstract: Phycocyanin, derived from marine algae, is known to have noteworthy antineoplastic properties. However, the underlying mechanism involved in phycocyanin-mediated anti-growth function on non-small cell lung cancer (NSCLC) cells is still ambiguous. Here, we investigated the mechanism of action of phycocyanin on H1299, A549, and LTEP-a2 cells. According to the results obtained, substrate 1 (IRS-1) expression was reduced by phycocyanin. Cell tests showed that siRNA knockdown of IRS-1 expression significantly inhibited the growth, migration, colony formation, but promoted the apoptosis of NSCLC cells. Meanwhile, phycocyanin and IRS-1 siRNA treatment both reduced the PI3K-AKT activities in NSCLC cells.   Moreover, overexpression of IRS-1 accelerated the proliferation, colony formation, and migration rate of H1299, A549, and LTEP-a2 cells, which was contradicting to the knockdown results. Overall, Citation: Hao, S.; Li, Q.; Liu, Y.; Li, F.; this study uncovered a regulatory mechanism by which phycocyanin inhibited the growth of NSCLC Yang, Q.; Wang, J.; Wang, C. Insulin cells via IRS-1/AKT pathway, laying the foundation for the potential target treatment of NSCLC. Receptor Substrate 1 Is Involved in the Phycocyanin-Mediated Keywords: phycocyanin; non-small cell lung cancer (NSCLC); proliferation; migration; insulin Antineoplastic Function of Non-Small receptor substrate 1 (IRS-1); antineoplastic Cell Lung Cancer Cells. Molecules 2021, 26, 4711. https://doi.org/ 10.3390/molecules26164711

Academic Editor: Assunta Pandolfi 1. Introduction The incidence and mortality rate of lung cancer is increasing at an alarming rate world- Received: 2 July 2021 wide [1]. Worldwide, an estimated 19.3 million new cancer cases and almost 10.0 million Accepted: 31 July 2021 cancer deaths occurred in 2020 [2]. Lung cancer is the leading cause of cancer death (18%) Published: 4 August 2021 and the second commonly diagnosed cancer (11.4%) in the past year [2–4]. Similar to most malignant tumors, lung cancer is composed of cell subpopulations with different molecular Publisher’s Note: MDPI stays neutral characteristics, leading to heterogeneity within the tumor [5]. Non-small cell lung cancer with regard to jurisdictional claims in (NSCLC), one of the subtypes of lung cancer, takes up approximately 85% of lung cancer published maps and institutional affil- cases [6], with characteristics of such as metastasis, low cure rate, and high mortality [7]. In iations. recent years, substantial progresses have been made in the emergence of various targeted therapies and the effective application of immunotherapy on NSCLC [8]. However, the main challenges still remain, such as how to identify new driver to expand the population benefiting from targeted therapy, and how to search for better predictors or Copyright: © 2021 by the authors. novel functional factors for NSCLC therapies [9]. Therefore, continuous drug development Licensee MDPI, Basel, Switzerland. provides increasing patients with the hope of prolonging survival. This article is an open access article The insulin receptor substrate (IRS) family is cytoplasmic adaptor , which distributed under the terms and play important roles as downstream signaling intermediates of cell surface receptors, and conditions of the Creative Commons many of which have been reported related to cancers [10]. The IRS cascades are the Attribution (CC BY) license (https:// most valuable protein cascades in the insulin signaling pathway, including IRS-1, IRS-2, creativecommons.org/licenses/by/ IRS-3, IRS-4, IRS-5, and IRS-6 [11], of which, IRS-1 and IRS-4 are most associated with 4.0/).

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tumor growth and proliferation, and IRS-2 is the most frequently associated with tumor movement and invasion [12]. IRS-1, one of the cytoplasmic adaptor proteins in IRS family, is found in insulin-stimulated Fao liver cancer cells for the first time [10], which was derived from insulin-stimulated rat liver [13] and 3T3-L1 cells [14]. It is reported that the expression of IRS-1 is increased in hepatocellular [15], pancreatic [16], and [17], while its expression is negatively correlated with tumor progression in breast [18] cancer. The specific expression and function of IRS-1 in human cancer has aroused our continuous attention to follow-up trials. Phycocyanin, a type of natural blue pigment, mainly exists in Spirulina and is used as dietary supplement in many countries [19]. Recently, phycocyanin has attracted great attention due to its non-toxic and functional characteristics. The therapeutic value of phyco- cyanin has been widely confirmed [20] including antioxidant [21], anti-inflammatory [22], anticancer [23], hypolipidemic [24], neuroprotective [25], and immunomodulatory ef- fects [26]. In particular, phycocyanin was proved to exert anti-cancer effect on a variety of cancer types such as [27], liver cancer [28], NSCLC [29], and colon cancer [30] in vivo and in vitro. Strikingly, although the antineoplastic function of phycocyanin on NSCLC has been widely studied, the study mainly focused on the phenotype analysis. The underlying inhibitory mechanism of phycocyanin in NSCLC is still required further investigation. In this work, the anticancer mechanism of phycocyanin in NSCLC was analyzed based on H1299, A549, and LTEP-a-2 NSCLC cells. The results were expected to lay a theoretical foundation for the future treatment of NSCLC and the utilization of phycocyanin.

2. Results 2.1. IRS-1 Expression Is Downregulated by Phycocyanin in NSCLC Previously, we have demonstrated that phycocyanin (4.8 µM) exerted significantly antineoplastic function on multiple NSCLC cell lines including A549 (Figure S1), H1299, and LTEP-a2 cells [31]. A RNA-seq method was performed to further explore the mech- anism of phycocyanin on A549 cell line. A strict comparison at adjusted p ≤ 0.05 and log2FC fold change ≥1 (for upregulation) or ≤−1 (for downregulation), was made to determine the differentially expressed genes (DEGs) between phycocyanin-treated (4.8 µM) and untreated groups. The list of DEGs is presented in Supplementary Table S1. In total, 2970 genes are significantly differentially expressed, including 1431 downregulated and 1539 upregulated genes. IRS-1 (marked in yellow, line 888 in Table S1), one of the differen- tially expressed genes, is significantly downregulated (0.61 ± 0.21) compared with control group (1.32 ± 0.15, Figure1A). To verify whether phycocyanin could reduce the expression of IRS-1 in NSCLC cells, we performed qRT-PCR and Western blot assay. As shown in Figure1B,C, the expressions of IRS-1 significantly decreased after phycocyanin treatment in A549, H1299, and LTEP-a2 cells, which was consistent with transcriptome results. These results suggested that IRS-1 might participate in the antineoplastic function of phycocyanin in NSCLC cells. Molecules 2021, 26, x FOR PEER REVIEW 3 of 12 Molecules 2021, 26, 4711 3 of 11

Figure 1. IRS-1 expression is downregulated by phycocyanin in NSCLC. (A) mRNA-seq results of IRS-1 expression in Figure 1. IRS-1 expression is downregulated by phycocyanin in NSCLC. (A) mRNA-seq results of A549 cells after 4.8 µM phycocyanin treatment. (B,C) qRT-PCR and Western blot analysis of IRS-1 expressions after 4.8 µM phycocyaninIRS-1 expression treatment in in A549 A549, H1299,cells after and LTEP-a24.8 μM cells.phycocyanin PC, phycocyanin. treatment * p <. 0.05; (B,C **) pqRT< 0.01.-PCR and Western blot analysis of IRS-1 expressions after 4.8 μM phycocyanin treatment in A549, H1299, and LTEP-a2 cells. PC, phycocyanin.2.2. Knockdown * p of< 0 IRS-1.05; ** Expression p < 0.01. Promotes Apoptosis of NSCLC Cells The functions of the ectopic expression of IRS-1 on cell phenotype were explored 2.2. Knockdown of IRSusing-1 Expression siRNA transfection. Promotes The Apoptosis silencing of efficacy NSCLC of IRS-1 Cells was validated by qRT-PCR and Western blot analysis (Figure2A). As a result, compared with control groups, the expres- The functions sionsof the of IRS-1ectopic were expression significantly of decreased IRS-1 on after cell siRNA phenotype transfection were in A549, explored H1299, and using siRNA transfection.LTEP-a2 The cells, silencing indicating efficacy that IRS-1 of expression IRS-1 was was validated successfully by silenced. qRT-PCR As and shown in Western blot analysisFigure (Figure2B , siRNA 2A). knockdown As a result, of IRS-1 compared expression with significantly control promoted groups, the the proportion ex- of pressions of IRS-1 werelate apoptotic significantly A549 (5.26% decreased± 0.37%), after H1299 siRNA (5.24% transfection± 0.12%), and LTEP-a2in A549, (4.18% H1299± 0.04%, ) cells, respectively. In addition, the proportion of early apoptotic A549 cells was also sig- and LTEP-a2 cells, indicating that IRS-1 expression was successfully silenced. As shown nificantly induced (3.72% ± 0.14%) after IRS-1 silencing. It is worth noting that although in Figure 2B, siRNAthe knockdown late apoptotic of rates IRS are-1 expression increased in RRS-1significantly knockdown promoted cells, they the are propor- far less than tion of late apoptoticthat A549 in phycocyanin-treated (5.26% ± 0.37%), H1299 H1299 (11.30% (5.24%± 0.16%)± 0.12%) and, LTEP-a2and LTEP (14.50%-a2 (4.1± 0.68%)8% ± cells. 0.04%) cells, respectively.This indicates In addition, that phycocyaninexerts the proportion antineoplastic of early apoptotic effects of NSCLC A549 cells cells via was complex also significantly inducedmechanisms, (3.72 and% ± IRS-1 0.14%) might after be just IRS one-1 silencing of the potential. It is regulatory worth noting factors that of phyco- cyanin. Subsequently, the expressions of apoptosis markers were analyzed at protein levels although the late apoptotic(Figure2C) rates. It was are shown increased that siRNA in RRS knockdown-1 knockdown of IRS-1 cell coulds, they significantly are far less promote than that in phycocyaninthe expressions-treated of H1299 pro-apoptotic (11.30 % ± Bax,0.16%) whereas and slightly LTEP- reducea2 (14.50 the expressions% ± 0.68%) of anti- cells. This indicatesapoptotic that phycocyaninexerts gene Bcl-2. These results antineoplastic indicated that theeffects ectopic of expression NSCLC ofcells IRS-1 via affected complex mechanisms,the apoptosisand IRS- of1 might A549, H1299, be just and one LTEP-a2 of the cells, potentia whichl mightregulatory be one factors of the potential of mechanisms of phycocyanin on NSCLC. phycocyanin. Subsequently, the expressions of apoptosis markers were analyzed at pro- tein levels (Figure 2C). It was shown that siRNA knockdown of IRS-1 could significantly promote the expressions of pro-apoptotic gene Bax, whereas slightly reduce the expres- sions of anti-apoptotic gene Bcl-2. These results indicated that the ectopic expression of IRS-1 affected the apoptosis of A549, H1299, and LTEP-a2 cells, which might be one of the potential mechanisms of phycocyanin on NSCLC.

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Figure 2. Knockdown of IRS-1 expression promotes apoptosis of NSCLC cells. (A) The silencing efficacy detection of Figure 2. Knockdown of IRS-1 expression promotes apoptosis of NSCLC cells. (A) The silencing efficacy detection of IRS-1 transcription (24 h) and protein (48 h) levels after 50 nM siRNA transfection in A549, H1299, and LTEP-a2 cells. IRS-1 transcription (24 h) and protein (48 h) levels after 50 nM siRNA transfection in A549, H1299, and LTEP-a2 cells. (B) (ApoptosisB) Apoptosis analysis analysis of A549, of A549, H1299 H1299,, and and LTEP LTEP-a2-a2 cells cells after after 50 nM 50 nMsiRNA siRNA knockdown knockdown of IRS of- IRS-11 expressions expressions for 48 for h 48. (C h.) (WesternC) Western blot blot analysis analysis of ofBax Bax and and Bcl Bcl-2-2 expressions expressions after after 50 50 nM nM IRS IRS-1-1 siRNA siRNA transfection transfection in in A549, A549, H1299 and LTEP-a2LTEP-a2 cellscells forfor 4848 h.h. ** pp << 0.05;0.05; ** p < 0 0.01..01.

2.3.2.3. Knockdown of IRS-1IRS-1 Expression Inhibits the Proliferation,Proliferation, Migration,Migration, andand AKTAKT ActivityActivity ofof NSCLC Cells The cellcell growthgrowth curve curve showed showed that that siRNA siRNA knockdown knockdown of IRS-1 of IRS expression-1 expression significantly signifi- inhibitedcantly inhibited the proliferation the proliferation abilities ofabilities A549, H1299,of A549, and H1299 LTEP-a2, and cells LTEPin vitro-a2 cells (Figure in vitro3A) . Besides,(Figure 3A). the colonyBesides, forming the colony abilities forming of NSCLC abilities cells of NSCLC were also cells suppressed were also suppressed after IRS-1 siRNAafter IRS treatment-1 siRNA (Figuretreatment3B), (Figure suggesting 3B), suggesting that IRS-1 that influences IRS-1 influences the unanchored the unanchored growth abilitygrowth of ability NSCLC of cells.NSCLC The cells. wound-healing The wound assay-healing showed assay that showed silencing that IRS-1 silencing expression IRS-1 significantlyexpression significantly reduced the reduced migration the abilities migration of NSCLCabilities cellsof NSCLC at 24 and cells 48 at h, 24 respectively and 48 h, (Figurerespectively3C). The (Figure PI3K-AKT 3C). The signaling PI3K- wasAKT involved signaling in was multiple involved regulatory in multiple processes regulatory includ- ingprocesses cell proliferation, including cell migration proliferation, and apoptosis migration [32 an]. Tod apoptosi explores whether [32]. To phycocyanin explore whether and IRS-1phycocyanin regulate and PI3K-AKT IRS-1 regulate pathway PI3K in- NSCLCAKT pathway cells, we in furtherNSCLC analyzed cells, we thefurther phosphoryla- analyzed tionthe phosphorylation levels of related levels proteins. of related As shown proteins. in Figure As shown3D,E, phycocyaninin Figure D and and E, IRS-1 phycocyanin siRNA treatmentand IRS-1 bothsiRNA reduced treatment the phosphorylationboth reduced the levels phosphorylation of AKT proteins levels in of A549, AKT H1299, proteins and in

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LTEP-a2A549, H1299 cells,, indicatingand LTEP- phycocyanina2 cells, indicating might inhibitphycocyanin proliferation might andinhibit migration proliferation of NSCLC and cellsmigration through of NSCLC downregulating cells through IRS-1/AKT downreg activities.ulating IRS-1/AKT activities.

FigureFigure 3.3.Knockdown Knockdown ofof IRS-1IRS-1 expression expression inhibits inhibits the the proliferation, proliferation, migration migration and and AKT AKT activity activity of of NSCLC NSCLC cells. cells. (A ()A Prolif-) Pro- erationliferation assay assay of NSCLCof NSCLC cells cells after after50 nM50 nMIRS-1 IRS siRNA-1 siRNA treatment. treatment. (B) Colony(B) Colony formation formation assay assay of NSCLC of NSCLC cells cells after after 50 nM 50 IRS-1nM IRS siRNA-1 siRNA treatment. treatment. (C) Wound-healing (C) Wound-healing assay assay of NSCLC of NSCLC cells cells after after 50 nM 50 IRS-1 nM IR siRNAS-1 siRNA treatment. treatment. (D,E )(D Western,E) Western blot assayblot assay of PI3K-AKT of PI3K-AKT signaling signaling proteins proteins of NSCLC of NSCLC after after 4.8 µ4.8M μM phycocyanin phycocyanin and and 50 nM50 nM IRS-1 IRS siRNA-1 siRNA treatment treatment for for 48 48 h, h, respectively. PC, phycocyanin. * p < 0.05; ** p < 0.01. respectively. PC, phycocyanin. * p < 0.05; ** p < 0.01.

2.4.2.4. OverexpressionOverexpression ofof IRS-1IRS-1 PromotesPromotes ProliferationProliferation andand MigrationMigration ofof NSCLCNSCLC CellsCells ToTo furtherfurther verifyverify thethe regulatoryregulatory functionfunction ofof IRS-1IRS-1 inin NSCLCNSCLC cells,cells, wewe constructedconstructed PCMV7.1-IRS-1PCMV7.1-IRS-1 vectorvector (data (data not not shown) shown) and and performed performed IRS-1 IRS overexpression-1 overexpression experiments. experi- Here,ments. as Here, IRS-1 a overexpressions IRS-1 overexpression had little had effect li onttle LTEP-a2effect on cell LTEP phenotype,-a2 cell ph theenotype, phenotype the resultsphenotype were results shown were only shown in A549 only and in H1299A549 and cells. H1299 Figure cells4A. showedFigure 4A the showed expressions the ex- ofpressions IRS-1 in of A549 IRS- and1 in H1299A549 and cells H1299 after PCMV7. cells after 1-IRS-1 PCMV7. vector 1-IRS transfection.-1 vector transfection. Compared withCompared control with groups, control the transcriptiongroups, the transcriptio and proteinn levelsand protein of IRS-1 levels significantly of IRS-1 upregulatedsignificantly afterupregulated plasmid after transfection. plasmid transfection. Proliferation Proliferation analysis showed analysis that showed overexpression that overexpres- of IRS- 1sion could of IRS promote-1 could the promote growth the ability growth of A549ability cells, of A549 while cells, it hadwhile small it had (but small significant) (but sig- effectnificant) on H1299effect on proliferation H1299 proliferatio (Figure4B).n (Figure However, 4B). increasing However, the increasing IRS-1 expression the IRS- both1 ex- promotedpression both the colonypromoted forming the colony abilities forming of A549 abilities and H1299 of A549 cells, and indicating H1299 cells, that IRS-1indicating was morethat IRS likely-1 was to affecting more likely the unanchoredto affecting the growth unanchored ability of growth NSCLC ability cells (Figureof NSCLC4C). cells As expected,(Figure 4C). overexpression As expected, of overexpres IRS-1 also significantlysion of IRS-1 accelerated also significantly the migration accelerated ratio ofthe A549 mi- andgration H1299 ratio cells of A549 (Figure and4D), H1299 which cells was (Figure in contrary 4D), which to the was siRNA in contrary knockdown to the results. siRNA Notably,knockdown although results. IRS-1 Notably expression, although is vast IRS at-1 bothexpression mRNA is and vast protein at both levels mRNA after and vector pro- transfection,tein levels after the vector observed transfection effects are, the rather observed small effects at the are phenotypic rather small level. at the This phenotypic might be duelevel to. This cell phenotypicmight be due variation to cell phenotypic depending variation on changes depending of multiple on changes factors, excessivelyof multiple enhancingfactors, excessive the expressionly enhanc ofing one the protein expression may of not one have protein an obvious may not effect. have Meanwhile,an obvious

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effect. Meanwhile, we also analyzed the effects of phycocyanin on IRS-1 overexpression

A549 and H1299 cellswe (Figure also analyzed S2). theThe effects results of phycocyaninshowed that on IRS-1phycocyanin overexpression could A549 signifi- and H1299 cantly reduce the proliferationcells (Figure S2).of IRS The-1 resultsoverexpression showed that A549 phycocyanin and H1299 could cells significantly. Taken reduce to- the gether, these resultsproliferation showed that of IRS-1 overexpression overexpression A549of IRS and-1 H1299promotes cells. Takenproliferation together, these and results migration of NSCLCshowed cells, that suggesting overexpression IRS of-1 IRS-1 played promotes an proliferationimportant androle migration in phycocya- of NSCLC cells, nin-mediated proliferationsuggesting and IRS-1 migration played aninhibition important process role in phycocyanin-mediated of NSCLC cells. proliferation and migration inhibition process of NSCLC cells.

FigureFigure 4. 4.Overexpression Overexpression of IRS-1 of promotes IRS-1 promotes proliferation proliferation and migration ofand NSCLC migration cells. (A )of Validation NSCLC of cells. IRS-1 transcription(A) Vali- (24dation h) and of protein IRS-1 (48 transcription h) expressions (24 after h) PCMV7.1-IRS-1 and protein vector (48 transfectionh) expressions in A549 after and H1299PCMV7.1 cells.-IRS (B) Proliferation-1 vector C analysistransfection of A549 in and A549 H1299 and cells H1299 after IRS-1cells.overexpression. (B) Proliferation ( ) Colonyanalysis forming of A549 analysis and ofH1299 A549 andcells H1299 after cells IRS after-1 IRS-1 overexpression. (D) Wound-healing analysis of A549 and H1299 migration after IRS-1 overexpression. * p < 0.05; overexpression. (C) Colony forming analysis of A549 and H1299 cells after IRS-1 overexpression. ** p < 0.01. (D) Wound-healing analysis of A549 and H1299 migration after IRS-1 overexpression. * p < 0.05; ** p < 0.01. 3. Discussion In this study, we investigated the underlying anticancer mechanism of phycocyanin, 3. Discussion a natural pigment mainly exists in Cyanobacteria, Rhodophyta, Cryptophyta, and Glau- In this study, wecophyta investigated [33]. Due the to underlying the safety features anticancer (no side mechanism effects on normal of phycocyanin, tissue cells) of phy- cocyanin [19], its antineoplastic ability in lung cancer is of particular interest. A large a natural pigment numbermainly of exists studies in have Cyanobacteria, shown that phycocyanin Rhodophyta, can inhibit Cryptophyta the proliferation,, and cause cell Glaucophyta [33]. Duecycle to arrest the andsafety induce features apoptosis (no of NSCLCside effects cells [34 on,35 ].normal Previously, tissue our teamcells had) of discov- phycocyanin [19], itsered antineoplastic that phycocyanin ability exerted in lung anti-proliferative cancer is of particular functions through interest. reducing A large receptor- number of studies haveinteracting shown serine/threonine-protein that phycocyanin can kinase inhibit 1 (RIPK1) the proliferation, and toll/interleukin cause cell 1 receptor domain-containing adaptor (TIRAP), two mediated proteins in multiple NSCLC cells, cycle arrest and inducerevealing apoptosis the potential of NSCLC anticancer cells mechanism [34,35]. Previously, of phycocyanin our in team NSCLC had [36 dis-,37]. Here, covered that phycocyaninIRS-1, a exerted key regulatory anti-proliferative protein of phycocyanin, functions was through investigated reducing to participate recep- in the tor-interacting serine/threoninegrowth and migration-protein processes kinase of1 (RIPK1) NSCLC cells. and toll/interleukin 1 receptor domain-containing adaptorAs a matter(TIRAP), of fact, two the IRSmediated families haveproteins been consideredin multiple as important NSCLC signal cells, interme- revealing the potentialdiates anticancer in insulin-regulated mechanism glucose of homeostasis, phycocyanin and playin NSCLC important [3 roles6,37 in]. tissueHere, growth and tumorigenesis [38]. It has been reported that IRS-1 contributes to the excessive growth IRS-1, a key regulatorystimulation protein in humanof phycocyanin, pancreatic cancer was [16 investigated]. Moreover, Dearth to participate et al. discovered in transgenicthe growth and migrationmice processes overexpressing of NSCLC IRS-1 in cells. the mammary gland showed progressive mammary hyperpla- As a matter of fact, the IRS families have been considered as important signal in- termediates in insulin-regulated glucose homeostasis, and play important roles in tissue growth and tumorigenesis [38]. It has been reported that IRS-1 contributes to the exces- sive growth stimulation in human pancreatic cancer [16]. Moreover, Dearth et al. dis- covered transgenic mice overexpressing IRS-1 in the mammary gland showed progres- sive mammary hyperplasia, tumorigenesis, and metastasis [39], which further suggested the oncogenicity of IRS-1. In this work, knockdown of IRS-1 expression resulted in the reduced proliferation and migration of H1299, A549, and LTEP-a2 cells, indicating IRS-1

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sia, tumorigenesis, and metastasis [39], which further suggested the oncogenicity of IRS-1. In this work, knockdown of IRS-1 expression resulted in the reduced proliferation and migration of H1299, A549, and LTEP-a2 cells, indicating IRS-1 was involved in regulating the growth of NSCLC cells, which was partially in accord with Dearth’s study. Interestingly, Han et al. reported that 43.6% of NSCLC specimens showed loss of IRS-1 expression, and downregulation of IRS-1 is more frequently observed in squamous cell carcinoma than other type lung cancer [40]. Meanwhile, low IRS-1 cytoplasmic expression also showed a trend toward association with decreased overall survival in squamous cell carcinoma patients [41]. In this study, IRS-1 was relatively highly expressed in A549, H1299, and LTEP-a2 cells, which might be due to these NSCLC cells being cells. The PI3K/AKT signaling pathway is indispensable for cell differentiation, and it has been evolutionarily conserved to maintain appropriate cell growth, survival, and [42]. IRS-1 is one of the central mediators in transmitting signals from IGF-IR receptors via the PI3K/AKT pathway and it is commonly overexpressed in cancer [43]. Meanwhile, Kathryn E et al. reported that inhibition of mTOR in tumors caused activation of Akt kinase which is associated with induction of IRS-1 and was prevented by IGF-IR inhibition [44]. Strikingly, Gokhan et al. [45] discovered that gain of function mutations of IRS-1 could be related to development of lung cancer, because IRS-1 binds to PI3Kp85 to activate Akt kinase. In the present study, we found that knockdown the expression of IRS-1 could inhibit the activity of PI3K-AKT signaling pathway. Xu et al. showed that PI3K-AKT activity could be inhibited by targeting IRS-1 in [46], which supported our results. Overall, this work explored the potential anti-growth mechanism of phycocyanin in NSCLC, and discovered that phycocyanin could inhibit the growth and migration, and promote apoptosis of NSCLC cells via downregulating IRS1 expression.

4. Materials and Methods 4.1. Cell Lines and Culture Condition Phycocyanin (isolated and purified from Spirulinaplatensis) standard substance was purchased from BinMei Biotechnology (Taizhou, China). Human NSCLC A549, H1299, and LTEP-a-2 cell lines were purchased from American Type Cell Collection (ATCC, Man- ◦ assas, VA, USA). Cells were maintained at 37 C in an atmosphere containing 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) supplemented by 10% fetal bovine serum (FBS), 0.1 mg/mL of streptomycin, and 100 units/mL of penicillin.

4.2. RNA-Seq Analysis The RNA-seq analysis was performed using HiSeq 4000 (Illumina, San Diego, CA, USA). Briefly, total RNA was extracted using Trizol after phycocyanin treatment (4.8 µM). Five micrograms of total RNA was used for transcriptome analysis. Base calling was adopted to convert original sequencing images to sequential data. The sequence and gene annotations were obtained from the UCSC Genome Website (http: //genome.ucsc.edu/, accessed on 14 June 2020). The differentially expressed genes be- tween phycocyanin-treatment and control cells were identified based on fragments per kilobase million reads (FPKM). The control and phycocyanin-treated groups were analyzed in triplicate.

4.3. Cell Proliferation Assay Cell proliferation assay was detected by the MTT method [31]. Briefly, cells (40% cell density) were added into 96-well plate and treated with 4.8 µM phycocyanin for 24 h. 100 µL 10% MTT medium was added to the experimental cells, followed by 100 µL SDS- HCL lysate addition after 4 h at 37 ◦C. The absorbance value (570 nm) was observed after 15 h of culture. Molecules 2021, 26, 4711 8 of 11

4.4. Cell Colony Formation Assay Cell colony formation assay was performed according to pervious work [31]. Briefly, cells in the exponential growth phase were harvested and transferred to a six-well plate (20% cell density), followed by 4.8 µM phycocyanin treatment for 10–15 days. The number of cell clones was counted after Giemsa staining.

4.5. Cell Cycle Assay Cells in the exponential growth phase were treated by 4.8 µM phycocyanin for 48 h, then harvested and fixed by 70% cold ethanol for at least 24 h, then resuspended in a 500 µL of propidium iodide (PI)/RNase staining buffer, followed by washing with cold PBS. Cell cycle distribution was measured using FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA).

4.6. Cell Apoptosis Assay Cells in the exponential growth phase were treated by 4.8 µM phycocyanin for 48 h, then harvested and resuspended gently in 100 µL binding buffer, followed by adding 5 µL of AnnexinV and 7AAD staining solution, respectively, for 20 min. Non-apoptotic cells cannot be stained by AnnexinV and 7AAD (Annexin V−/7AAD−). Annexin V could bind the cellular phosphotidylserine, which would expose to the surface of cells in early apoptotic process (Annexin V+/7AAD−). 7AAD could bind the nucleic acid of late apoptotic or death cells (Annexin V+/7AAD+). The apoptosis of cells was analyzed by FACS Calibur (Becton Dickinson).

4.7. siRNA Transfection Cells were added into 6-well plate (50% cell density) and cultured at 37 ◦C for 12 h. 50 nM of siRNAs were transfected into cells according to the manufacturer’s instructions (Dharmacon, Lafayette, CO, USA). Negative siRNA was used as the negative control.

4.8. Wound-Healing Assay Cells in the exponential growth phase were harvested and transferred to 6-well plates until the confluence was 100%. The cell “wound” was scratched by the sterile tips in 6-well plates after 4.8 µM phycocyanin treated. Photos of the wounds were taken every 12 h, and the widths of the wounds were measured at three positions for each replicate using the Leica Application Suite (Leica Microsystems GmbH, Wetzlar, Germany).

4.9. Quantitative Real-Time PCR (qRT-PCR) Analysis Total RNA of cells was extracted with Trizol reagent, followed by reverse tran- scription with primescript RT master mix. qRT-PCR analysis was performed using Ap- plied Biosystems Step One-Plus (Waltham, MA, USA) and SYBR Green real-time PCR Master Mix Kit (Takara, Dalian, China). GAPDH was used as an endogenous control. The primers were as follows: IRS-1 up 50-TACTACGAGAACGAGAAG-30 and down 50- TTGTTGATGTTGAAGCAG-30. GAPDH up 50-AGGGCTGCTTTTAACTCT-30 and down 50-TGGGTGGAATCATATTGGA-30. The relative expression of each gene was calculated and normalized using the 2−∆∆Ct method relative to GAPDH. Each assay was performed in quadruplicate. Three independent experiments were carried out.

4.10. Western Blot Analysis Total protein was extracted from cell by RIPA lysis buffer (1% NP40, 0.1% SDS, 5 mM EDTA, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate) containing 0.1 µM pro- tease and phosphatase inhibitors, and centrifuged at 12,000 r for 45 min at 4 ◦C. Protein concentrations were measured using the Bradford method. The equivalent amounts of pro- teins (50 µg) were separated by 12% SDS-PAGE and then transferred to a polyvinylidened- ifluoride (PVDF) membrane. Primary antibodies including anti-IRS-1 (1:500), anti-Bax (1:1000), anti-Bcl-2 (1:1000), anti-p-PTEN (1:500), anti-p-PDK1 (1:1000), anti-p-AKT Ser473 Molecules 2021, 26, 4711 9 of 11

(1:500), anti-p-AKT Thr308 (1:1000), anti-C-Raf (1:500), and anti-GSK-3β (1:1000) were incubated with the membrane at 4 ◦C overnight, followed by washing with PBS for 15 min. Secondary antibody anti-β-actin (1:5000) was then incubated for 30 min at 37 ◦C and wash the followed by signal detection using an electrochemical luminescence (ECL) system.

4.11. Statistical Analysis The numerical data were expressed as means ± standard deviation (SD). Two-tailed Student’s t-test was performed for comparison among the different groups. In addition, p < 0.05 (*) or p < 0.01 (**) was considered as statistically significant.

Supplementary Materials: The following are available online. Table S1: Detailed information of differentially expressed genes (DEGs). Figure S1: Inhibitory effects of phycocyanin (4.8 µM) on A549 cells. Figure S2: Proliferation analysis of A549 and H1299 cells after IRS-1 overexpression and PC treatment. Author Contributions: Conceptualization, S.H. and C.W.; Funding acquisition, S.H.; Investigation, Q.L., Y.L., F.L., Q.Y. and J.W.; Validation, Q.L., F.L., Q.Y. and J.W.; Writing—original draft, Y.L.; Writing—review and editing, S.H. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Natural Science Foundation of China, grant number 32072231; The Excellent youth cultivation program of BTBU (19008021150). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing is not applicable. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of phycocyanin are available from the authors.

References 1. Dubey, A.K.; Gupta, U.; Jain, S. Epidemiology of lung cancer and approaches for its prediction: A systematic review and analysis. Chin. J. Cancer 2016, 35, 71. [CrossRef] 2. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021, 71, 209–249. [CrossRef] 3. Siegel, R.L.; Miller, K.D.; Fuchs, H.E.; Jemal, A. Cancer Statistics, 2021. CA Cancer J. Clin. 2021, 71, 7–33. [CrossRef] 4. Hirsch, F.R.; Scagliotti, G.V.; Mulshine, J.L.; Kwon, R.; Curran, W.J.; Wu, Y.L.; Paz-Ares, L. Lung cancer: Current therapies and new targeted treatments. Lancet 2017, 389, 299–311. [CrossRef] 5. Zhang, J.; Fujimoto, J.; Zhang, J.; Wedge, D.C.; Song, X.; Zhang, J.; Seth, S.; Chow, C.-W.; Cao, Y.; Gumbs, C.; et al. Intratumor heterogeneity in localized lung delineated by multiregion sequencing. Science 2014, 346, 256–259. [CrossRef] 6. Ettinger, D.S.; Wood, D.E.; Akerley, W.; Bazhenova, L.A.; Borghaei, H.; Camidge, D.R.; Cheney, R.T.; Chirieac, L.R.; D’Amico, T.A.; Dilling, T.J.; et al. NCCN Guidelines Insights: Non-Small Cell Lung Cancer, Version 4.2016 Featured Updates to the NCCN Guidelines. J. Natl. Compr. Canc. Netw. 2016, 14, 255–264. [CrossRef][PubMed] 7. Molina, J.R.; Yang, P.; Cassivi, S.D.; Schild, S.E.; Adjei, A.A. Non-small cell lung cancer: Epidemiology, risk factors, treatment, and survivorship. Mayo Clin. Proc. 2008, 83, 584–594. [CrossRef] 8. Carlisle, J.W.; Ramalingam, S.S. A banner year for immunotherapy and targeted therapy. Nat. Rev. Clin. Oncol. 2019, 16, 79–80. [CrossRef][PubMed] 9. Herbst, R.S.; Morgensztern, D.; Boshoff, C. The biology and management of non-small cell lung cancer. Nature 2018, 553, 446–454. [CrossRef][PubMed] 10. White, M.F.; Maron, R.; Kahn, C.R. Insulin rapidly stimulates phosphorylation of a Mr-185,000 protein in intact cells. Nature 1985, 318, 183–186. [CrossRef] 11. Chakraborty, C.; Agoramoorthy, G.; Hsu, M.J. Exploring the evolutionary relationship of insulin receptor substrate family using computational biology. PLoS ONE 2011, 6, e16580. [CrossRef] 12. Mardilovich, K.; Pankratz, S.L.; Shaw, L.M. Expression and function of the insulin receptor substrate proteins in cancer. Cell Commun. Signal. 2009, 7, 1–15. [CrossRef] 13. Rothenberg, P.L.; Lane, W.S.; Karasik, A.; Backer, J.; White, M.; Kahn, C.R. Purification and partial sequence analysis of pp185, the major cellular substrate of the insulin receptor . J. Biol. Chem. 1991, 266, 8302–8311. [CrossRef] Molecules 2021, 26, 4711 10 of 11

14. Keller, S.R.; Kitagawa, K.; Aebersold, R.; Lienhard, G.E.; Garner, C.W. Isolation and characterization of the 160,000-Da phosphoty- rosyl protein, a putative participant in insulin signaling. J. Biol. Chem. 1991, 266, 12817–12820. [CrossRef] 15. Boissan, M.; Beurel, E.; Wendum, D.; Rey, C.; Lécluse, Y.; Housset, C.; Lacombe, M.-L.; Desbois-Mouthon, C. Overexpression of insulin receptor substrate-2 in human and murine . Am. J. Pathol. 2005, 167, 869–877. [CrossRef] 16. Bergmann, U.; Funatomi, H.; Kornmann, M.; Beger, H.G.; Korc, M. Increased expression of insulin receptor substrate-1 in human pancreatic cancer. Biochem. Biophys. Res. Commun. 1996, 220, 886–890. [CrossRef][PubMed] 17. Hellawell, G.O.; Turner, G.D.H.; Davies, D.R.; Poulsom, R.; Brewster, S.F.; Macaulay, V.M. Expression of the type 1 insulin-like growth factor receptor is up-regulated in primary prostate cancer and commonly persists in metastatic disease. Cancer Res. 2002, 62, 2942–2950. 18. Schnarr, B.; Strunz, K.; Ohsam, J.; Benner, A.; Wacker, J.; Mayer, D. Down-regulation of insulin-like growth factor-I receptor and insulin receptor substrate-1 expression in advanced human breast cancer. Int. J. Cancer 2000, 89, 506–513. [CrossRef] 19. Kuddus, M.; Singh, P.; Thomas, G.; Al-Hazimi, A. Recent developments in production and biotechnological applications of C-phycocyanin. Biomed. Res. Int. 2013, 2013, 742859. [CrossRef][PubMed] 20. Manirafasha, E.; Ndikubwimana, T.; Zeng, X.; Lu, Y.; Jing, K. Phycobiliprotein: Potential microalgae derived pharmaceutical and biological reagent. Biochem. Eng. J. 2016, 109, 282–296. [CrossRef] 21. Benedetti, S.; Benvenuti, F.; Pagliarani, S.; Francogli, S.; Scoglio, S.; Canestrari, F. Antioxidant properties of a novel phycocyanin extract from the blue-green alga Aphanizomenonflos-aquae. Life Sci. 2004, 75, 2353–2362. [CrossRef] 22. Cherng, S.-C.; Cheng, S.-N.; Tarn, A.; Chou, T.-C. Anti-inflammatory activity of c-phycocyanin in lipopolysaccharide-stimulated RAW 264.7 macrophages. Life Sci. 2007, 81, 1431–1435. [CrossRef] 23. Gantar, M.; Dhandayuthapani, S.; Rathinavelu, A. Phycocyanin induces apoptosis and enhances the effect of topotecan on prostate cell line LNCaP. J. Med. Food 2012, 15, 1091–1095. [CrossRef] 24. Han, L.-K.; Li, D.-X.; Xiang, L.; Gong, X.-J.; Kondo, Y.; Suzuki, I.; Okuda, H. Isolation of pancreatic lipase activity-inhibitory component of spirulinaplatensis and it reduce postprandial triacylglycerolemi. YakugakuZasshi. 2006, 126, 43–49. [CrossRef] 25. Pentón-Rol, G.; Marín-Prida, J.; Pardo-Andreu, G.; Martínez-Sánchez, G.; Acosta-Medina, E.F.; Valdivia-Acosta, A.; Lagumersindez-Denis, N.; Rodríguez-Jiménez, E.; Llópiz-Arzuaga, A.; López-Saura, P.A.; et al. C-Phycocyanin is neu- roprotective against global cerebral ischemia/reperfusion injury in gerbils. Brain Res. Bull. 2011, 86, 42–52. [CrossRef] 26. Chen, H.-W.; Yang, T.-S.; Chen, M.-J.; Chang, Y.-C.; Wang, E.I.-C.; Ho, C.-L.; Lai, Y.-J.; Yu, C.-C.; Chou, J.-C.; Chao, L.K.-P.; et al. Purification and immunomodulating activity of C-phycocyanin from Spirulinaplatensis cultured using power plant flue gas. Process Biochem. 2014, 49, 1337–1344. [CrossRef] 27. Li, B.; Chu, X.; Gao, M.; Li, W. Apoptotic mechanism of MCF-7 breast cells in vivo and in vitro induced by photodynamic therapy with C-phycocyanin. Acta Biochim. Biophys. Sin. 2010, 42, 80–89. [CrossRef] 28. Roy, K.R.; Arunasree, K.M.; Reddy, N.P.; Dheeraj, B.; Reddy, G.V.; Reddanna, P. Alteration of mitochondrial membrane potential by Spirulinaplatensis C-phycocyanin induces apoptosis in the doxorubicinresistant human hepatocellular-carcinoma cell line HepG2. Biotechnol. Appl. Biochem. 2007, 47, 159–167. [CrossRef] 29. Bingula, R.; Dupuis, C.; Pichon, C.; Berthon, J.-Y.; Filaire, M.; Pigeon, L.; Filaire, E. Study of the Effects of Betaine and/or C-Phycocyanin on the Growth of Lung Cancer A549 Cells In Vitro and In Vivo. J. Oncol. 2016, 2016, 8162952. [CrossRef] 30. Wang, H.; Liu, Y.; Gao, X.; Carter, C.L.; Liu, Z.-R. The recombinant beta subunit of C-phycocyanin inhibits cell proliferation and induces apoptosis. Cancer Lett. 2007, 247, 150–158. [CrossRef][PubMed] 31. Hao, S.; Yan, Y.; Li, S.; Zhao, L.; Zhang, C.; Liu, L.; Wang, C. The In Vitro Anti-Tumor Activity of Phycocyanin against Non-Small Cell Lung Cancer Cells. Mar. Drugs 2018, 16, 178. [CrossRef] 32. Lin, L.; Li, Q.; Hao, W.; Zhang, Y.; Zhao, L.; Han, W. Upregulation of LncRNA Malat1 Induced Proliferation and Migration of Airway Smooth Muscle Cells via miR-150-eIF4E/Akt Signaling. Front. Physiol. 2019, 10, 1337. [CrossRef] 33. Yu, P.; Wu, Y.; Wang, G.; Jia, T.; Zhang, Y. Purification and bioactivities of phycocyanin. Crit. Rev. Food Sci. Nutr. 2017, 57, 3840–3849. [CrossRef] 34. Baudelet, P.-H.; Gagez, A.-L.; Bérard, J.-B.; Juin, C.; Bridiau, N.; Kaas, R.; Thiéry, V.; Cadoret, J.-P.; Picot, L. Antiproliferative activity of Cyanophoraparadoxa pigments in melanoma, breast and lung cancer cells. Mar. Drugs 2013, 11, 4390–4406. [CrossRef] 35. Li, B.; Gao, M.-H.; Chu, X.-M.; Teng, L.; Lv, C.-Y.; Yang, P.; Yin, Q.-F. The synergistic antitumor effects of all-trans retinoic acid and C-phycocyanin on the lung cancer A549 cells in vitro and in vivo. Eur. J. Pharmacol. 2015, 749, 107–114. [CrossRef][PubMed] 36. Hao, S.; Li, S.; Wang, J.; Zhao, L.; Yan, Y.; Wu, T.; Zhang, J.; Wang, C. C-Phycocyanin Suppresses the In Vitro Proliferation and Migration of Non-Small-Cell Lung Cancer Cells through Reduction of RIPK1/NF-κB Activity. Mar. Drugs 2019, 17, 362. [CrossRef][PubMed] 37. Hao, S.; Li, S.; Wang, J.; Yan, Y.; Ai, X.; Zhang, J.; Ren, Y.; Wu, T.; Liu, L.; Wang, C. Phycocyanin Exerts Anti-Proliferative Effects through Down-Regulating TIRAP/NF-κB Activity in Human Non-Small Cell Lung Cancer Cells. Cells 2019, 8, 588. [CrossRef] [PubMed] 38. Shaw, L.M. The insulin receptor substrate (IRS) proteins: At the intersection of metabolism and cancer. Cell Cycle 2011, 10, 1750–1756. [CrossRef][PubMed] 39. Dearth, R.K.; Cui, X.; Kim, H.-J.; Kuiatse, I.; Lawrence, N.A.; Zhang, X.; Divisova, J.; Britton, O.L.; Mohsin, S.; Allred, D.C.; et al. Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2. Mol. Cell. Biol. 2006, 26, 9302–9314. [CrossRef][PubMed] Molecules 2021, 26, 4711 11 of 11

40. Han, C.H.; Cho, J.Y.; Moon, J.T.; Kim, H.J.; Kim, S.K.; Shin, D.H.; Chang, J.; Ahn, C.M.; Kim, S.K.; Chang, Y.S. Clinical significance of insulin receptor substrate-I down-regulation in non-small cell lung cancer. Oncol. Rep. 2006, 16, 1205–1210. [CrossRef] 41. Piper, A.J.; Clark, J.L.; Mercado-Matos, J.; Matthew-Onabanjo, A.N.; Hsieh, C.C.; Akalin, A.; Shaw, L.M. Insulin Receptor Substrate-1 (IRS-1) and IRS-2 expression levels are associated with prognosis in non-small cell lung cancer (NSCLC). PLoS ONE 2019, 14, e0220567. [CrossRef][PubMed] 42. Courtnay, R.; Ngo, D.C.; Malik, N.; Ververis, K.; Tortorella, S.M.; Karagiannis, T.C. Cancer metabolism and the Warburg effect: The role of HIF-1 and PI3K. Mol. Biol. Rep. 2015, 42, 841–851. [CrossRef][PubMed] 43. Barker, R.M.; Holly, J.M.P.; Biernacka, K.M.; Allen-Birt, S.J.; Perks, C.M. Mini Review: Opposing Pathologies in Cancer and Alzheimer’s Disease: Does the PI3K/Akt Pathway Provide Clues? Front. Endocrinol. 2020, 11, 403. [CrossRef][PubMed] 44. O’Reilly, K.E.; Rojo, F.; She, Q.B.; Solit, D.; Mills, G.B.; Smith, D.; Lane, H.; Hofmann, F.; Hicklin, D.J.; Ludwig, D.L.; et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res. 2006, 66, 1500–1508. [CrossRef] [PubMed] 45. Gorgisen, G.; Hapil, F.Z.; Yilmaz, O.; Cetin, Z.; Pehlivanoglu, S.; Ozbudak, I.H.; Erdogan, A.; Ozes, O.N. Identification of novel mutations of insulin receptor substrate 1 (IRS1) in tumor samples of non-small cell lung cancer (NSCLC): Implications for aberrant insulin signaling in development of cancer. Genet. Mol. Biol. 2019, 42, 15–25. [CrossRef][PubMed] 46. Xu, Y.; Chen, J.; Gao, C.; Zhu, D.; Xu, X.; Wu, C.; Jiang, J. MicroRNA-497 inhibits tumor growth through targeting insulin receptor substrate 1 in colorectal cancer. Oncol. Lett. 2017, 14, 6379–6386. [CrossRef][PubMed]