Table 1.

In vitro studies on synthetic PI3K/Akt/mTOR inhibitors in PC.

Treatment	Cell Line Name	Mechanism of Action	Results	Reference
Everolimus (0.5–5 nM) and Torkinib (PP242) (0.5–5 nM)	DU145 & 22RV1 PC	Everolimus: mTORC1 inhibition PP242: dual mTORC1/mTORC2 inhibition	↓cell growth ↓proliferation rates	[62]
Rapamycin (10 nM)	PC3, MDCK, and COS-7	mTORC1 inhibition	Prostate tumor overexpressed -1, stimulates protein translation in an mTORC1-dependent manner	[63]
Rapamycin (250 nM); Torin-1 (250 nM); and Cycloheximide (10 µg/mL)	MEFs (WT(4EBP1/2+/+; p53−/−) and DKO (4EBP1/2−/−; p53−/−)	Torin-1: ATP-competitive inhibitors of mTORC1 Torin-1 blocked canonical mTORC1-dependent events (phosphorylation of S6K1 and 4E-BP1), ↓35S-Cys/Met incorporation, ↓translation of eIF4B, and ↓cytoplasmic ribosomal proteins Rapamycin: ↓mTORC1 kinase activity	4E-BPs mediate acute mTOR inhibition ↓translation of TOP and TOP-like mRNAs TOP mRNAs require eIF4G1 to anchor eIF4E to the cap →translational regulation by the 4E-BPs and mTORC1	[64]
Rapamycin (0.1–1000 nM) and p-XSC (0.625–10 μM)	LNCaP, C4-2, and DU145	↓p-XSC →Akt expression (by inhibition of mTORC2) Rapamycin: ↓mTORC1 kinase activity	↓cell growth and viability ↓phosphorylation of the mTORC2 and mTORC1 downstream targets, Akt, RPS6, phospho-RPS6 (Ser235/236), and PKCα ↓IC50 values p-XSC→↓levels of Rictor, Combination more effectively inhibited viability and mTOR signaling	[65]
Rapamycin (20 ng/mL)	PC3	Inhibition of mTORC1	↓translation in normoxic cells and 2-2-under hypoxic conditions	[66]
Rapamycin (100 nM)	PC3 and LNCaP PC	Rapamycin: inhibitor of mTOR activity	↓NF-κB DNA-binding activity ↓IKK activity ↓RelA/p65phosphorylation→ blocked ↓S6K phosphorylation	[67]
LY294002 (10 nM) and Rapamycin (100 nM)	PC3 and LNCaP	LY294002:PI3-kinase antagonist Rapamycin: inhibitor of mTOR activity	↓NF-κB-dependent reporter activity ↓RelA/p65phosphorylation	[67]
Bicalutamide (10 µM–50 μM) and Ridaforolimus (0.5 Nm–50 nM)	LNCaP and C4-2	Ridaforolimus: ↓mTOR signaling, ↑p-Akt levels, and ↓phospho-S6 levels Bicalutamide: AR blockade	↑cell cycle arrest ↓S and G2/M phases and G1 arrest Ridaforolimus→↑PSA expression Bicalutamide→↓Ridaforolimus-induced stimulation of PSA ↓cell proliferation	[68]
Ridaforolimus (0.0001–1000 nM)	LNCaP and C4 (PTEN−/−)	Ridaforolimus: ↓mTOR signaling, ↑p-Akt levels, ↓phospho-S6 levels	↓cellular proliferation	[68]
Bortezomib (10, 100 nM) (in hypoxic or normoxic condition)	LNCaP and PC3	↓PI3K/Akt/mTOR and MAPK pathways ↓p44/42 MAPK phosphorylation	↓HIF-1α levels transcriptional activity and protein expression ↓phosphorylation of the downstream targets of Akt, p70S6K(Thr389), and S6RP(Ser235/236) (hypoxic condition) ↓nuclear HIF-1α levels ↑ cytoplasmic HIF-1α (hypoxic condition) ↓baseline VEGF secretion levels ↓proteasome activity protein levels of total Akt26 and total amount of p44/42 MAPK →no change	[69]
Rapamycin (20 nM) and Torin-1 [(PC3 & DU145 (250 nM), LNCaP (125 nM)]	DU145, LNCaP, and PC3	Torin-1→↓ phosphorylation of pro-ADM1	AMD1 mRNA expression: In PC3 with Torin, was less than Rapamycin. In LNCaP with Rapamycin, was more than Torin. In DU145 with Rapamycin, was more than Torin.	[70]
Torin-1 (250 nM) and Torin-1 (250 nM) + MG132 (5 µM)	DU145 cells expressing Myc-AMD1-HA	Torin-1→ mTORC1 inhibition→↓proAMD1 stability	↓half-life of proAMD1	[70]
Afrocyclamin A (50 pM); Wortmannin (30 nM) + afrocyclamin A; LY294002 (20 mM) + afrocyclamin A; Rapamycin (100 nM) + afrocyclamin A; and Actinomycin D (5 mg/mL) + afrocyclamin A	LN1	Rapamycin and Actinomycin D: ↓p70S6K→↓mTORC1-Akt pathway signaling LY294002 and Wortmannin: inhibition of PI3K→↓p-Akt→↓PI3K/Akt/mTOR signaling	↓pS6-Kinase and 4EBP1 ↓PSA synthesis and cell proliferation	[71]
Venuloside A (50 μM)	LNCaP-FGC and PC3	ESK242 and ESK246: ↓p70S6K→↓mTORC1 activity	↓Leucine uptake ↓cell viability in LNCaP and PC3 (only ESK242) downregulation of CDK1 and UBE2C	[72]
Salinomycin (4 µM)	PC3 and DU145	↓p70S6K & pS6→↓activation of mTORC1	↓LRP6 expression and phosphorylation ↓axin2 expression ↓Wnt/βcatenin signaling ↓ colony formation ↓ cell growth ↓cancer cells viability	[42]
Dactolisib (0.5–1.5 µM) and Rapamycin (100 nM)	UMN and 4240P	BEZ235: ↓activation of 4EBP1 and 70S6K→↓mTORC1-Akt pathway signaling Rapamycin: ↓only p70S6K→↓mTORC1-Akt pathway signaling	↓HK2 protein expression	[25]
Sapanisertib (100 nM)	PC3 and LNCaP	Blocked of mTORC 1/2 and downregulation of mTOR regulator genes→ ↓mTORC1/2 activation	Downregulation of cyclin D1 and HIF 1α/2α ↑apoptosis no effect on androgen receptor	[34]
3- carboxymethylpyrrolidine -2,4-dicarboxylic acid (2 µmol/L)	PC3 and DU145	↓ phosphorylation of NF-κB and IκBα ↑cellular caspase-3/7 activity ↓colony formation ↑cell apoptosis ↓expression of survivin ↑expression of cleaved caspase-3	↓cancer cells growth ↓proliferation	[73]
LY294002 (10 μM)	PC3	↓phosphorylation of Akt→↓mTOR activity	↓pTSC2, pS6K, and p65 No effects on expression of pr regulation of NF-κB through mTORC1	[24]
Rapamycin (100 nM) + IKKβ inhibitor (5 μM)	TSC2 null	Rapamycin: ↓p70S6K→↓mTORC1-Akt pathway signaling	↓mTORC1 activity ↑NF-κB phosphorylation ↓phosphorylation of mTORC1 ↓IRS1	[24]
Rapamycin (1 nM) and Bicalutamide (10 μM)	LNCaP and C4-2	Expression of Akt inhibited apoptosis induced by combination of rapamycin and Bicalutamide mediated by mTORC1	Induction of apoptosis in C4-2 cells by inhibition of both Raptor and Rictor Bicalutamide→ ↑Raptor inhibition Overexpression of both Raptor and Rictor → inhibition of apoptosis induced by Rapamycin and Bicalutamide mTORC1 inhibited while mTORC2 stimulated AR activity and Akt phosphorylation ↑AR transcriptional activity in Rapamycin-treated cells by active form of Akt (pCMV-6-myr-Akt-HA) mTORC1 and AR activate parallel cell survival pathways	[74]
Flutamide (40–70 µg/mL) + temsirolimus (CCI-779) (0.01–800 nM)	PTEN−/−TP53−/− stem/progenitor prostate epithelial cells	mTOR and AR signaling are required clonogenic and have tumor-initiating activity in PTEN−/−TP53−/− prostate progenitor cells	↓tumor growth and size	[75]
8-CPT-2Me-cAMP (100, 150 µM); LY294004 (20 µM); and Rapamycin (100 µM)	1-LN	Expression of Akt and mTOR inhibited	CPT-2Me-cAMP (100 µM): ↑ levels of Raptor and Rictor ↑ levels of p-PRAS40 and GbL induction of Epac1 signaling →activation of mTORC1 and mTORC2 CPT-2Me-cAMP (150 µM): upregulation of p-AktT308 and p-AktS437 expression ↑sensitivity to LY294002 LY294002: suppression of 8-CPT-2MecAMP- dependent p-AktS473 and p-AktS473 kinase activities	[76]
Salinomycin (50 nM)	AR-expressing LNCaP (castration-sensitive) and C4-2B (castration resistant) PC cells	↓phosphorylation of RPS→↓activity of mTORC1	↓cytostasis ↓apoptosis ↓ autophagy ↓AR mRNA and protein levels	[37]
Fingolimod (a derivative of FTY720 or SPS-7) (10 µM)	LNCaP and DU-145	↓phosphorylation of p70S6K, Akt and 4EBP1→↓Akt/mTOR signaling pathway	↓apoptosis ↓cell growth ↓ cell cycle in G1	[77]
Fenofibrate (50 μM)	PC3	↓phosphorylation of p70S6K→↓phosphorylation of mTOR	↓aroliferation ↓apoptosis ↑AMPK and MAPK phosphorylation ↓cell growth	[78]
CORM-2 (40 µM)	LNCaP and PCa	Expression of LKB1→ ↑AMP/AMT radio→↓mTOR	↓apoptosis ↓proliferation ↑active form of caspase	[79]
mTOR shRNA-expressing lentivirus (LV-shmTOR) (20–40 μg)+ vector-derived LV as control (LV-shCON) (20–40 μg)	RWPE1, LNCap, and C4-2b	↓Expression of Akt and mTOR	↓proliferation in prostate cancer cells in mTOR shRNA-transduced cells ↓Akt, PI3K, S6K, and 4EBP1 (inC4-2b cells)	[80]
Panobinostat (10 nM) + dactolisib (BEZ235) (500 nM)	PC3 and PC3-AR	BEZ235-induced inhibition of downstream targets of mTORC1, p-4EBP1, and p-S6K ↓p-Akt, p-ATM & ↑apoptosis	Upregulation of ATM-Akt-Erk1/2 signaling → antitumor activity	[81]
VS-5584 (SB2343) (4–1000 nM)	PC3 (with PTEN deletion) MV4-11 cells (FLT3-ITD)	In PC3 cells: inhibition of both PI3K and mTOR-signaling cascade In MV4-11 cells: blocked of pAkt(S473) and pAkt (T308)	The enzyme inhibitory properties of VS-5584 translates into the modulation of the PI3K/mTOR-signaling pathway	[82]
Nitroxoline (1–30 μM)	PC3 and LNCaP	AMPK phosphorylation → activity of TSC1/TSC2 complex → mTOR inhibition →↓cell proliferation LC3-II protein →cell apoptosis	↑AMPKα phosphorylation ↓mTOR phosphorylation ↑LC3-II protein	[83]
NSK-01105 (a sorafenib derivative) (10 μmol/L) + sorafenib (10 μmol/L)	LNCaP and PC3	↓EGFR activity→ blocking the Raf/MEK/ERK and Akt/mTOR pathway	↓Raf-1 kinase activity ↓EGFR phosphorylation by NSK-01105 ↓MEK and ERK phosphorylation in both treatments ↓phosphorylation of Akt by NSK-01105	[84]
Voxtalisib (XL765) (5 μM) + pilaralisib (XL147) (10 μM) + rapamycin (1 µM)	PC3, LNCaP, C-81, C4-2B, 22rv1, DU145, and everolimus-resistant PC3 cells	PI3K/Akt pathway→ cell proliferation AKT/mTORC1/4EBP1 signaling pathway→ tumor cell growth	↓phosphorylation of Akt and PDK1 ↓4EBP1 phosphorylation ↓cell proliferation ↑ apoptosis	[85]
Diaminobenzidine (1.7–35.8 ng/mL)	PC3, DU145, LNCaP, and PNT1A	↓ amounts of RICTOR and RAPTORs ↓ mTOR activity	↓phosphorylation of Akt and 4E-BP1 No change in pS6K and pSG upregulation of mLST8	[34]
Salinomycin (50–400 nM)	RWPE-1, LNCaP, and C4-2	Salinomycin →inhibiting AR Salinomycin →inhibiting the PI3K/Akt/mTORC1 pathway	↓cell proliferation ↑sensitivity Salinomycin (200 nM): ↓RWPE-1 Salinomycin (200 nM): ↓castration-resistant in C4-2 400 nM Salinomycin: ↓RWPE-1 less than C4-2 ↑autophagy Robust cleavage of PARP1 and procaspase-3 in C4-2 ↓phosphorylation of mTOR ↓phospho-AR in LNCaP	[37]
Remotiflori radix (100–500 mg/mL)	PC3 and DU145	↑ cells in G1, ↓ cells in S, and G2/M→ cell death	↑ cell death	[86]
Remotiflori radix (250, 500 mg/mL)	PC3, DU145, and HT1080	LC3 expression → ↓ autophagy YO-PRO-1 uptake → early cell apoptosis AMPK phosphorylation → ULK1 protein kinase activation→ ↓ mTORC1 activity → cell death	↓LC3 expression ↑YO-PRO-1 uptake ↑cleaved caspases and PARP cleavage ↑AMPK phosphorylation ↓mTORC1 activity	[86]
Metformin (1585 μg/mL); Vitamin D3 (400 μg/mL); and Metformin (1585 μg/mL) + Vitamin D3(400 μg/mL)	DU145	G1/S cell cycle arrest → cell death ↑ AMPK phosphorylation → inhibition of the mTOR/S6K signaling pathway → ↓ cell proliferation	↓proliferation Combination of Metformin (1585 μg/mL) and vitamin D3 (400 μg/mL) Reduced proliferation rate	[24]
CCL2 (100 ng/mL)	PC3	CCL2 treatment→AMPK phosphorylation→mTORC1 signaling inhibition and autophagy induction	Induction of mTORC1 activation and downregulation of AMPK/Raptor phosphorylation in serum-starved PC3 cells → cell survival AMPK directly phosphorylates Raptor on Ser792 to inhibit mTORC1 D942(AMPK activator) induces Raptor phosphorylation and downregulates mTORC1 signaling, promoting cell death in PC3 but CCL2 reverses the lethal effect of D942	[55]
Rapamycin (1 µM)	SH-SY5Y	Down-regulation of PINK1 by siRNA and treatment with rapamycin reduced the number of migrated cells, and if PINK1 activates mTORC2, overexpression of PINK1 may affect cell motility	PINK1 induces phosphorylation of Rictor → activation of mTORC2 → stimulation of cell motility	[87]
Docetaxel (5 nM); Everolimus (0.5–5 nM); and Everolimus (0.5–5 nM) + docetaxel (5 nM)	PC3	mTOR induction→↑ SK1 expression→↑ VEGF→↑ P70S6K phosphorylation	Docetaxel: no change in P70S6K phosphorylation and SK1 mRNA↑ VEGF mRNA Everolimus alone or with docetaxel: ↓P70S6K phosphorylation, ↓SK1 mRNA,↓ VEGF expression and secretion	[61]
7-aza-tetrahydroquinazoline (140 nm)	NCI-PC3 (PTEN-null)	Inhibit both mTORC1 and mTORC2	Compound 12 h→the most potent compounds 12a-h→ potency and selectivity for mTOR over PI3Kα and PI3Kδ	[88]
7-aza-tetrahydroquinazoline (140 nm)	NCI-PC3 (PTEN-null)	Inhibit both mTORC1 and mTORC2	Selectivity for PI3Kα↑ ↑interaction with Trp2239 in mTOR ↑antiproliferative	[88]

Abbreviations: ↓ = decrease; ↑ = increase; → = leads to; AMD1 = adenosylmethionine decarboxylase-1; AMPK = 5′-AMP-activated protein kinase; CCL2 = (C-C motif) ligand 2; CORM-2 = carbon monoxide–releasing molecule-2; 8-CPT-Me-cAMP = 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate; IKKβ = IκB kinase β; LY294002 = 2-(morpholin-4-yl)-8-phenyl-4H-1-benzopyran-4-one; MEKK3 = mitogen-activated protein kinase kinase kinase 3; MG132 = carbobenzoxy-Leu-Leu-leucinal; PI3K = phosphatidylinositol 3-kinase; PINK1 = PTEN-induced kinase 1; p-XSC = p-xyleneselenocyanate; SK1 = sphingosine-kinase-1; TOP = terminal oligopyrimidine; and VEGF = vascular endothelial growth factor.