The Impact of Oxidative Stress and AKT Pathway on Cancer Cell Functions and Its Application to Natural Products

Oxidative stress and AKT serine-threonine kinase (AKT) are responsible for regulating several cell functions of cancer cells. Several natural products modulate both oxidative stress and AKT for anticancer effects. However, the impact of natural product-modulating oxidative stress and AKT on cell functions lacks systemic understanding. Notably, the contribution of regulating cell functions by AKT downstream effectors is not yet well integrated. This review explores the role of oxidative stress and AKT pathway (AKT/AKT effectors) on ten cell functions, including apoptosis, autophagy, endoplasmic reticulum stress, mitochondrial morphogenesis, ferroptosis, necroptosis, DNA damage response, senescence, migration, and cell-cycle progression. The impact of oxidative stress and AKT are connected to these cell functions through cell function mediators. Moreover, the AKT effectors related to cell functions are integrated. Based on this rationale, natural products with the modulating abilities for oxidative stress and AKT pathway exhibit the potential to regulate these cell functions, but some were rarely reported, particularly for AKT effectors. This review sheds light on understanding the roles of oxidative stress and AKT pathway in regulating cell functions, providing future directions for natural products in cancer treatment.


Introduction
The AKT (AKT serine-threonine kinase; protein kinase B; PKB) pathway, which consists of AKT and AKT downstream effectors, is involved in regulating many cell functions such as cell survival, proliferation, metabolism [1], angiogenesis, and migration [2], by activating AKT [3,4]. AKT is commonly overexpressed in several kinds of cancer [5].
AKT activity is modulated by phosphorylation and dephosphorylation for activation and inactivation [6]. AKT is activated through several routes, mainly by ligand-receptor tyrosine kinase phosphorylation that activates phosphoinositide 3-kinase (PI3K) and consequently AKT [7]. Growth factors and cytokines are common ligands for AKT activation [8]. Furthermore, AKT is also activated by cellular stressors, such as heat shock [9], ultraviolet irradiation [10], and hypoxia [11].
Oxidative stress is the status where cells exhibit higher reactive oxygen species (ROS) levels than antioxidants, causing an imbalance of redox homeostasis [14,15]. ROS include non-radical and radical chemical species. Examples of non-radical ROS include organic hydroperoxides (ROOH), singlet molecular oxygen (O 2 ), electronically excited carbonyl, ozone (O 3 ), and hypochlorous and hypobromous acid (HOCl and HOBr). Examples of free-radical ROS include superoxide anion radical (O 2 ·−), hydroxyl radical (·OH), peroxyl radical (ROO·), and alkoxyl radical (RO·). ROS are generated as by-products of several cell functions, such as energy production. Moreover, ROS is also generated by exposure to drugs, toxins, and radiation [16].
Different treatments may show different responses to oxidative stress and AKT activation. Notably, the following examples from various cell lines demonstrate the potential for interaction between oxidative stress and AKT activation. This needs careful investigation in case other cell lines are concerned because their genetic mutations may differ. Four responses to oxidative stress and AKT, namely (1) oxidative stress activates AKT, (2) oxidative stress inhibits AKT, (3) AKT induces oxidative stress, and (4) AKT suppresses oxidative stress, were summarized as follows ( Figure 1). Natural products may generate oxidative stress [30] and modulate AKT expression [31,32] in cancer cell treatments. However, the potential regulation of cell functions by natural product-modulating oxidative stress and AKT pathway (AKT and AKT effectors) lacks systemic understanding. The modulating effects of natural products on oxidative stress and the AKT pathway will be discussed later.

Autophagy and Oxidative Stress
Autophagy is an intracellular catabolic process where long-lived proteins and dysfunctional organelles are degraded for recycling to generate energy under nutrient depletion or stress [94]. Oxidative stress controls autophagy in modulating cell survival and development [95,96]. The interaction between oxidative stress and autophagy in cancer cells has been reported from tumor initiation to cancer therapy [97].

ER Stress and Oxidative Stress
ER is a dynamic organelle involving several cellular functions [99,100]. Three primary unfolded protein response (UPR) mediators are identified, including protein kinase-RNAlike ER kinase (PERK), inositol-requiring enzyme 1 alpha (IRE1α), and activating transcription factor 6 (ATF6) [46] (Table 1). Under cell stress, the ER environment is unstable, protein maturation is dysfunctional, and misfolded protein accumulates, which triggers UPR to recover normal ER function by attenuating protein translation, enhancing degradation of misfolded proteins, and up-regulating molecular chaperones for protein folding [99,100].
Many drugs possess modulating effects of oxidative stress and ER stress [47] for cancer cell death. For example, curcumin analog WZ35 may induce oxidative stress-dependent ER stress and G2/M arrest, leading to cell death of prostate cancer cells [101]. Oxidative stress may regulate several mediators and affect ER stress ( Figure 1, Table 1). MAPK signaling can regulate ER stress response [48]. Consequently, ER stress can induce apoptosis via MAPK p38 and JNK [49].
Oxidative stress may regulate several mediators and affect mitochondrial morphogenesis ( Figure 1, Table 1), leading to apoptosis and cell death [109]. For example, ROS activates mitochondrial fission through DRP1 [50]. DRP1 interacts with FIS1 to cause mitochondrial fission and oxidative stress [51]. MFN2 is required for ROS production and inflammation in macrophages [110]. siMFN1 induces ROS generation of myoblast cells, which is suppressed by MFN1 overexpression [52]. Moreover, high glucose could cause oxidative stress in renal tubular epithelial cells and trigger mitochondrial fission and apoptosis [104]. Accordingly, inhibiting mitochondrial fission and enhancing mitochondrial fusion prevents apoptosis [111], whereas induction of mitochondrial fission promotes apoptosis [104].
For example, RETRA, the small molecule regulating the REactivation of Transcriptional Reporter Activity, can induce p53-associated gene expressions. RETRA promotes necroptosis in cervical cancer cells by phosphorylating RIPK1, RIPK3, and MLKL and inducing oxidative stress generation, which is reverted by necrostatin-1 [121]. Heat stress causes intestinal injury by up-regulating RIPK1/RIPK3 and inducing necroptosis, which is suppressed by oxidative stress scavenger N-acetylcysteine [122].
MAPK has a modulating ability for necroptosis ( Figure 1). For example, sulforaphane, a cruciferous vegetable-derived compound, suppresses necroptosis of microglia-mediated neuron damage by inhibiting MAPK expression [123]. Dimethyl fumarate, a drug for treating multiple sclerosis, promotes necroptosis of colon cancer cells by inducing oxidative stress and activating MAPK [124].

Senescence and Oxidative Stress
Cellular senescence arrests the cycling of damaged cell proliferation as a tumor suppressor mechanism. ROS induces senescence in several cell types [129,130]. For example, SIRT1, SIRT3, and SIRT6 may inhibit vascular senescence [71] (Table 1).
Several mediators of mitochondrial morphogenesis (fission and fusion) have been reported, such as DRP1, FIS1, MFN1, and MFN2 (Table 2). These mitochondrial morphogenesis mediators show a distinct modulation by PI3K/AKT/mTOR. Several examples of mitochondrial morphogenesis-modulating effects of PI3K/AKT/mTOR were described as follows.

DNA Damage Response and AKT
AKT also controls DNA damage responses such as DNA damage/repair ( Figure 1, Table 2). This concept was supported by several reports as follows. TH588 (a MutT homolog 1 (MTH1) inhibitor) and BKM120 (a pan-PI3K inhibitor) combined treatment promotes DNA damage and apoptosis by activating PI3K/AKT/mTOR in glioma cells [189]. Various PI3K isoforms differentially regulate the cell cycle, DNA replication, and DNA damage repair [190].
Moreover, combined with radiation, PKI-587, a dual PI3K/mTOR inhibitor, suppresses cell proliferation and tumor growth of liver cancer, accompanied by apoptosis. This PKI-587/radiation combined treatment inhibits PI3K/AKT/mTOR and homologous recombination (HR) repair-related kinases such as the ATM and the ATM and Rad3-related (ATR) in liver cancer cells (Table 2) [164]. PI3K signaling is overexpressed in ovarian cancer, contributing to chemoresistance, DNA replication, and genome stability. AKT improves DSB repair of non-homologous end joining (NHEJ) mediated by DNA-PK [191] (Table 2). Therefore, the inactivation of PI3K may inhibit DNA repair from improving ovarian cancer therapy [192].

Migration and AKT
AKT also modulates cell migration ( Figure 1). E2F2, a member of the E2F transcription factor family, is overexpressed in gastric cancer with poor overall survival. E2F2 overexpression in gastric cancer cells improves migration and invasion by down-regulating PI3K/AKT/mTOR-mediated autophagy [208].
Several mediators of migration have been reported, such as matrix metalloproteinase 2 (MMP2), MMP9, and EMT signaling ( Table 2). These migration mediators exhibit distinct modulation by PI3K/AKT/mTOR. Several examples of migration-modulating effects of PI3K/AKT/mTOR were earlier reported. AKT indicates an interaction relationship with MMP2 and MMP9 to modulate EMT [2,12]. Several examples demonstrate their interaction connecting to migration. Overexpression of Rab11a, a Rab GTPase, improves MMP2 expression and PI3K/AKT activation in promoting migration of liver cancer cells, which is suppressed by AKT inhibitor [209]. This suggests that AKT can activate MMP2 to improve the migration of cancer cells. PI3K/AKT can up-regulate MMP9 expression in limbal epithelial cells [210]. MMP9 activates the AKT/PI3K to trigger the EMT process, such as CDH1 down-regulation and CDH2 up-regulation, enhancing the proliferation and invasion of Wilms' tumor-derived cells [170]. Moreover, AKT activation also regulates other EMTrelated signaling proteins, such as VIM and snail (SNAI1) [171]. Therefore, AKT, MMP2, MMP9, and EMT-related signaling cooperatively modulate cell migration (Table 2).

Cell-Cycle Progression and AKT
The involvement of AKT-modulating AMPK, SIRT1, and MAPK in several functions was mentioned in Table 3. Notably, AMPK, SIRT1, and MAPK can regulate cell-cycle progression. The detailed regulation of the cell cycle was discussed in Section 2.10.

Apoptosis and AKT Effectors
The connection between AKT effectors and apoptosis is widely investigated in several studies (Table 4). For example, FOXO up-regulation triggers apoptosis of several cancer cells [236]. AKT down-regulation causes FOXO3a-mediated apoptosis of prostate cancer [237]. c-Myc can modulate cancer cell proliferation and apoptosis [253]. Glutaminolysis activates mTORC1 to suppress autophagy and trigger apoptosis of cancer cells [266]. Moreover, a high concentration of mTORC1 inhibitor (Everolimus) [332] triggers extrinsic apoptosis of colon cancer cells associated with inhibiting 4EBP1 [267]. Similarly, mTORC2 knockdown enhances apoptosis of breast cancer cells [268]. S6K1 and S6K2 have distinct functions in cancer cells [333]. S6K1 knockdown activates AKT to suppress apoptosis, while S6K2 knockdown inactivates AKT to promote apoptosis [281]. Additionally, inhibition of S6K1 by rosmarinic acid methyl ester (RAME) triggers apoptosis of cervical cancer cells [282]. It indicates that S6K1 and S6K2 can differentially regulate apoptosis in cancer cells. SREBP1 knockdown causes antiproliferation and triggers apoptosis in pancreatic cancer cells [292]. In contrast, SREBP1 overexpression induced by high glucose enhances proliferation and inhibits apoptosis of pancreatic cancer cells [293]. It indicates that SREBP1 can inhibit apoptosis in cancer cells.

Autophagy and AKT Effectors
The connection between AKT effectors and autophagy is widely investigated in several studies ( Table 4). The relationship between FOXO and autophagy in cancer cells was reviewed [238]. For example, SIRT1 improves FOXO1 deacetylation for inducing autophagy of cardiac myocytes by overexpressing RAB7A, member RAS oncogene family (RAB7A) for autophagosome-lysosome fusion [240]. Moreover, FOXO3 also shows autophagy-inducible function during muscle atrophy by enhancing several autophagy genes [239,334].

ER Stress and AKT Effectors
Several studies have widely investigated the connection between AKT effectors and ER stress (Table 4). For example, FOXO can interplay with ER stress in cancer [335]. FOXO1 inhibitor (AS1842856) triggers ER stress in unstimulated T cells [241]. FOXO inhibits nutrient restriction-induced ER stress of Tsc1 mutant cells [242]. It indicates that FOXO inhibits ER stress. Additionally, c-Myc causes ER stress by activating IRE1α-X-box binding protein 1 (XBP1) signaling [255]. c-Myc induces an adaptive ER stress in mice with liver tumor burden. In contrast, c-Myc knockdown down-regulates GRP78, ATF4, and CHOP [256]. It indicates that c-Myc induces ER stress.

Mitochondrial Morphogenesis and AKT Effectors
Several studies have widely investigated the connection between AKT effectors and mitochondrial fission/fusion (Table 4). For example, FOXO1 promotes MFN1 and MFN2 (fusion) expression but inhibits DRP1 and FIS1 (fission) expression, resulting in enlarged mitochondria of hepatocytes [243]. FOXO3 overexpression inhibits mitochondrial fission of cardiomyocytes by down-regulating MIEF2 [243]. However, FOXO3 may cause mitochondrial fission by activating Drp1 in phenylephrine-stimulated adult cardiomyocytes. It indicates that FOXO may regulate mitochondrial fission and fusion.

DNA Damage Response and AKT Effectors
Several studies have widely investigated the connection between AKT effectors and DNA damage response (Table 4). For example, N-methyl-N -nitro-N-nitrosoguanidine induces DNA damage in lung cancer cells by enhancing the nuclear import of FOXO1, which regulates DNA damage repair [246]. Moreover, FOXO3a suppresses genomic instability by inhibiting DNA double-strand break-induced mutations [247].

Migration and AKT Effectors
The connection between AKT effector and migration is widely investigated in several studies (Table 4). For example, FOXO is a mediator for regulating EMT expression [250]. FOXO3a suppresses the invasion of mammary adenocarcinoma cells and blocks TGF-β1promoted EMT in mouse mammary epithelial cells [251]. c-Myc knockdown inhibits cell migration of liver cancer cells [264]. mTORC1 inhibition suppresses hypoxia-induced migration of keratinocytes [280]. Knockdown of mTORC2 prevents cell migration [268].

Natural Products Targeting Apoptosis through Oxidative Stress
There are several oxidative stress-inducing natural products that modulate apoptosis ( Table 5). Cryptocarya-derived cryptocaryone triggers oxidative stress and promotes apoptosis of oral cancer cells [343]. Several Rubus fairholmianus-derived compounds stimulate oxidative stress and cause apoptosis of breast cancer cells [30]. Sanguinarine, a Sanguinaria canadensis-derived compound, is a bloodroot plant-derived natural alkaloid with antifungal effects [363]. The repurposing function of sanguinarine has been applied to trigger apoptosis for anticancer treatment. For example, sanguinarine inhibits thioredoxin reductase to induce oxidative stress and trigger apoptosis of cancer cells [344].

Natural Products Targeting Autophagy through Oxidative Stress
Several oxidative stress-inducible natural products are known that modulate autophagy (Table 5). Isoaaptamine, a marine sponge-derived compound, induces apoptosis and autophagy of breast cancer cells by generating oxidative stress [345]. Neferine, a Nelumbo nucifera-derived dibenzylisoquinoline alkaloid, induces the generation of ROS to trigger the autophagy of lung cancer cells [346]. Piperlongumine, a Piper longumderived compound, induces autophagy and cell death of osteosarcoma cells, reverted by N-acetylcysteine, suggesting that piperlongumine induces autophagy depending on oxidative stress [347].

Natural Products Targeting ER Stress through Oxidative Stress
Several anticancer drugs with oxidative stress-modulating ability can regulate ER stress (Table 5) [47]. For example, sarsasapogenin, an Anemarrhena asphodeloides-derived compound, promotes oxidative stress and ER stress in cervical cancer cells, reverted by N-acetylcysteine [348].

Natural Products Targeting DNA Damage Response through Oxidative Stress
Several oxidative stress-inducible natural products that enhance DNA damage response were provided as follows (Table 5). Cryptocaryone causes oxidative stress-dependent DNA damage in oral cancer cells [343]. Sinuleptolide, a soft corals-derived natural product, induces antiproliferation and oxidative stress in oral cancer cells, accompanied by DNA damage [356].

Natural Products Targeting Senescence through Oxidative Stress
Several oxidative stress-inducible natural products that modulate senescence were provided as following (Table 5). Apigenin inhibits oxidative stress-triggered senescence of lung fibroblasts [357]. Gingerenone A, a ginger-derived compound, inhibits proliferation and triggers oxidative stress and senescence of breast cancer cells, reverted by N-acetylcysteine [358].

Natural Products Targeting Migration through Oxidative Stress
Several oxidative stress-inducible natural products modulate migration (Table 5). Salinomycin promotes oxidative stress generation to decrease the proliferation and migration of prostate cancer cells [359]. Withaferin A blocks migration and invasion and induces oxidative stress of oral cancer cells, reverted by N-acetylcysteine [360].

Natural Products Targeting Apoptosis through AKT
Some natural products protect from apoptosis, but others induce it. Several reports investigated the apoptosis-protecting effects of natural products involving AKT (Table 6). For example, the suppression of oxidative stress and inflammation. Ginsenoside Rd, a Panax japonicus-derived natural product, enhances neural cell survival by reducing oxidative stress, increasing antioxidant expression, activating PI3K/AKT and ERK 1/2 pathways, and reducing apoptosis [377]. Troxerutin, a rutin-derived semi-synthetic bioflavonoid, decreases ROS and apoptosis by up-regulating antioxidant enzymes and translocating NRF2 [393]. Crocin, a crocus flower-derived carotenoid, suppresses retinal ischemia/reperfusion injurytriggered apoptosis of ganglion cells by activating AKT [369].

Natural Products Targeting Autophagy through AKT
Natural products may exhibit autophagy-modulating effects involving AKT (Table 6). For example, tanshinone IIA, a Salvia miltiorrhiza Bunge-derived bioactive compound, suppresses proliferation and induces autophagy of breast cancer cells by inactivating PI3K/AKT/mTOR signaling [411]. Patulin, a Penicillium-derived compound, promotes ROS generation and autophagy of liver cancer cells by inactivating AKT1/mTOR, reverted by N-acetylcysteine [408]. Fisetin exhibits a dual function for inhibiting PI3K/AKT and mTOR and promotes cytotoxic autophagy in prostate cancer cells [403]. Allicin, a bioactive compound from crushed garlic, triggers autophagy of liver cancer cells by inactivating AKT [397]. Crocin promotes autophagy and causes cell death of cervical cancer cells by activating AKT [400].
Moreover, allicin also suppresses 6-hydroxydopamine-up-regulated FIS1 and DRP1 expressions in pheochromocytoma PC12 cells, which trigger mitochondrial fission [449]. However, the role of AKT in mitochondrial fission-promoting effects of allicin was not reported by this study, and it warrants a detailed investigation in the future.

Natural Products Targeting Ferroptosis through AKT
The ferroptosis impact of natural products involving AKT was rarely investigated. Prostate [450] and lung [184] cancer cells highly express PI3K/AKT. Lung cancer cells inhibit ferroptosis by activating PI3K/AKT/mTOR [184]. In contrast, PI3K and mTOR inactivations induce ferroptosis in cancer cells [273]. Accordingly, it warrants a detailed investigation to identify AKT-modulating natural products in the future (Table 6).

Natural Products Targeting DNA Damage Response through AKT
Natural products may exhibit DNA damage response-modulating effects involving AKT (Table 6). For example, cerberin, a cardenolide isolated from the fruit kernel of Cerbera odollam, induces antiproliferation, anti-migration, apoptosis, ROS production, and DNA damage in cancer cells, accompanied by inhibiting PI3K/AKT/mTOR signaling [420]. Cucurbitacin-A, a cucurbitaceous plant-derived compound, exhibited antiproliferation, ROS generation, and DNA damage of ovarian cancer cells by inactivating PI3K/AKT/mTOR [422].

Natural Products Targeting Apoptosis through AKT effectors
Several studies investigated the connection between natural products and AKT effectorstriggered apoptosis (Table 7). For example, juglanthraquinone C, a Juglans mandshuricaderived compound, promotes ROS generation and triggers apoptosis of liver cancer cells by up-regulating FOXO signaling [455]. Dioscin, a natural steroidal saponin, triggers apoptosis of colon cancer cells by enhancing c-Myc ubiquitination [461].

Natural Products Targeting Mitochondrial Morphogenesis through AKT Effectors
Except for GSK3, above AKT effectors were rarely investigated with respect to natural products that target mitochondrial fission/fusion. Resveratrol suppresses 1-methyl-4-phenylpyridinium-induced mitochondrial fission of nigral dopaminergic cells by upregulating GSK3 expression [491].

Natural Products Targeting Necroptosis through AKT Effectors
AKT effectors provided by natural products that target necroptosis were rarely investigated.

Natural Products Targeting DNA Damage Response through AKT Effectors
Among these AKT effectors, only FOXO was reported to regulate DNA damage response by natural product treatments (Table 7). For example, purple corn extract reduces cigarette smoke-induced DNA damage to rodent blood cells by up-regulating FOX3a [457]. 6-Bromoindirubin-3 -oxime, a hemi-synthetic GSK3β inhibitor of indirubin derivative, inhibits DNA damage in fibroblasts [493].

Conclusions
Oxidative stress and the AKT pathway exhibit versatile effects in regulating cell function for cancer cell development and treatment. However, current information generally focuses on some of them without a comprehensive integration, particularly for AKT effectors such as FOXO, c-Myc, mTORC1/2, S6K1/2, SREBP1, 4EBP1, HIF, and GSK3.
As mentioned above, the impacts of oxidative stress and the AKT pathway (AKT and its effector) are well integrated into several cell functions such as apoptosis, autophagy, ER stress, mitochondrial fission/fusion, ferroptosis, necroptosis, DNA damage response, senescence, migration and cell-cycle progression.
In addition to establishing the connection between oxidative stress, AKT pathway, and cell functions, their impacts on cancer treatment by natural products are summarized and evaluated. Mounting literature evidence shows that several anticancer natural products regulate oxidative stress and AKT pathway. Accordingly, natural products with modulating effects on oxidative stress and AKT pathway are expected to provide the potential for cancer cell function regulation and impacts on cancer therapy. However, the contribution of oxidative stress and AKT pathway are as yet rarely connected to cell functions in anticancer treatments using natural products. After a detailed literature search, some cell functions are attributed to the modulating effects of oxidative stress and the AKT pathway, although some are not reported.
It should be noted that many reports for oxidative stress and AKT pathway-associated cell functions cited in this review are derived from investigations of several cancer cell lines. Since the genetic mutations for these cancer cell lines differ, the association between oxidative stress, AKT pathway, and some cell functions may be restricted or become dominant only in some cancer cells or unique treatments. This warrants a careful investigation of these relationships if different cancer cells are concerned.
We hypothesize that the involvement of oxidative stress and AKT pathway in natural product performance also provides potential anticancer impacts through the modulation of several cell functions (Figure 2). This review sheds light on the impacts of oxidative stress and AKT pathway-regulated cell functions, providing a better understanding and future directions for curing cancer with natural products.