J Clin Med Res

Journal of Clinical Medicine Research, ISSN 1918-3003 print, 1918-3011 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Clin Med Res and Elmer Press Inc

Journal website https://jocmr.elmerjournals.com

Original Article

Volume 17, Number 5, May 2025, pages 270-284

Anti-Breast Cancer Effects of Thymoquinone-Chemotherapeutic Combinations: A Systematic Review of the Latest In Vitro and In Vivo Studies

Figure 1. Flowchart illustrating the article selection procedure in accordance with the PRISMA 2020 guidelines for performing a systematic review.

**Table 1.** Characteristics and Main Outcomes of *In Vitro* Studies Included
Author (years)	Country	Intervention group	Control group	Cellular type	Carrier	Duration	Outcomes
ACNP: aragonite calcium carbonate (CaCO₃) nanoparticle; Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2; B-NE: borage nanoemulsion; Cis: cisplatin; CLNCs: chitosan-coated lipid nanocapsule; Cyclo: cyclophosphamide; DOX: doxorubicin; DTX: docetaxel; GCB: gemcitabine; 5-FU: 5-fluorouracil; LLCNs: lyotropic liquid crystalline nanoassemblies; LNCs: lipid nanocapsules; NE: nanoemulsion; p-5473-Akt: phosphorylated Akt; PLGA: poly lactic-co-glycolic acid; PPAR: peroxisome proliferator-activated receptor; PTEN: phosphatase and TENsin homolog; PTX: paclitaxel; TQ: thymoquinone; ULNCs: drug-loaded uncoated lipid nanocapsule.
Soni et al, 2015 [28]	India	TQ + PTX	PTX alone	MCF-7 breast cancer cell	PLGA nanoparticle	24 h	1) TQ + PTX significantly showed lower cell viability compared to PTX alone. P < 0.001. 2) TQ lowers the effective concentration (IC₅₀) of PTX. 3) TQ + PTX showed 0.688 (synergistic effect).
Sakalar et al, 2016 [29]	Turkey	TQ + PTX	PTX alone, untreated embryonic fibroblasts cell (PMEFCFL-P1)	4T1 (ATCC CRL-2539) breast cancer cells	-	48 h	1) TQ + PTX significantly showed lower cell viability compared to PTX alone. P < 0.0001. 2) Lower dose TQ showed higher apoptosis compared to untreated cells. P < 0.05. 3) TQ modulated apoptosis-related genes, cytokine, and p53 signaling pathway genes.
Bashmail et al, 2018 [40]	Saudi Arabia	TQ + GCB	GCB alone, untreated cell	MCF-7 and T47D breast cancer cells	-	24, 48, and 72 h	1) TQ lowers the effective concentration (IC₅₀) of GCB. 2) TQ + GCB showed 0.15 (strong synergistic effect). 3) TQ + GCB showed significantly higher apoptosis, increased cell death in the pre-G phase, and cell cycle arrest at S phase compared to GCB alone. P < 0.05. 4) TQ + GCB significantly increased autophagic cell death compared to control untreated cells. P < 0.05.
Zidan et al, 2018 [42]	Egypt	DOX + TQ + F2 gel, DOX + F2 gel, TQ + F2 gel	Free DOX, free TQ, untreated cell	MCF-7 breast cancer cells	F2 gel nanofibers	24 h	1) DOX + TQ + F2 gel showed lower cell viability compared to other treatments. P < 0.05. 2) DOX + TQ + F2 gel induced the highest apoptosis, 88.6%, compared to the control group.
Ibiyeye et al, 2019 [43]	Malaysia	DOX + TQ-ACNP, DOX-ACNP, TQ-ACNP	Free DOX + TQ, free TQ, free TQ	MDA-MB-231 breast cancer cell, MCF10A normal breast cell, 3T3 normal fibroblast cell	ACNP	24, 48, and 72 h	1) TQ + DOX showed lower cell viability than other treatments. P < 0.05. 2) Free TQ + DOX showed 0.8 at 48 h (slight synergism), while TQ + DOX-ACNP showed 0.5 at 48 h (synergism). 3) Free TQ increased the late apoptosis in TQ + DOX compared to DOX alone. 4) DOX + TQ-ACNP has the highest percentage in SubG0 (dead cells) and S phase at 48 h. 5) TQ has been shown to significantly inhibit cancer cell invasion and migration in TQ + DOX compared to other treatments. P < 0.05.
Khan et al, 2019 [44]	Saudi Arabia	TQ + cyclo	Cyclo alone, untreated cell	SKBR-3 and MDA-MB-231 breast cancer cells	-	48 h	1) TQ enhanced the growth inhibition of Her2⁺ and Her2^- breast cancer cells in combination with cyclo, inhibited p-5473-Akt, increased the expression of its inhibitor PTEN, and decreased cyclin D1 compared to cyclo alone. P < 0.001. 2) TQ + cyclo increased cells to arrest in sub-G1 and G1 compared to untreated cells. P < 0.05.
Odeh et al, 2019 [45]	Jordan	TQ + DTX	DTX alone	MCF-7 breast cancer cell	-	72 h	1) TQ lowers the effective concentration (IC₅₀) of DTX. 2) TQ + DTX showed 0.552 - 0.803 (synergistic effect).
Alkhatib et al, 2020 [46]	Saudi Arabia	TQ + DTX + B-NE	Free TQ and DTX	MCF-7 and MDA-MB-231 breast cancer cells	B-NE	48 h	1) TQ + DTX in B-NE exhibited a lower IC₅₀ than free DTX and other treatments. 2) TQ + DTX in B-NE showed 0.6-0.9 (synergistic effect). 3) TQ + DTX in B-NE significantly showed higher apoptosis compared to DTX alone. P < 0.05. 4) TQ + DTX and free TQ or DTX significantly showed higher autophagy than untreated cell. P < 0.05.
Bashmail et al, 2020 [47]	Saudi Arabia	TQ + PTX	PTX alone, TQ alone	MCF-7 and T47D breast cancer cells	-	24 and 48 h	1) TQ did not enhance PTX potency but significantly eliminated tumor-associated resistant cell clones to PTX. P < 0.05. 2) TQ + PTX showed 1.6 - 4.6 (antagonistic effect). 3) TQ + PTX significantly increased apoptosis, pre-G phase population, autophagic cell death fluorescence, and reduced CD44⁺/CD24^- compared to PTX alone. P < 0.05.
Bawadud et al, 2020 [48]	Saudi Arabia	NE-DTX + TQ, free DTX + TQ	Free DTX alone, free TQ alone, drug-free NEs	MCF-7 and MDA-MB-231 breast cancer cells	NE	48 h	1) DTX + TQ-NE and free DTX alone significantly increased DNA fragmentation compared to free TQ alone and drug-free NEs. P < 0.05. 2) DTX + TQ-NE significantly reduced CD44⁺/CD24^-, SNAIL-1, and TWIST-1 compared to other treatments. P < 0.05. 3) DTX + TQ-NE caused significant arrest at the G2/M phase (P < 0.0001) and the S phase (P < 0.0021) compared to untreated cells and free TQ alone.
Zafar et al, 2020 [30]	India	DTX + TQ in ULNCs and CLNCs	Free DTX alone, free TQ alone, DTX-LNCs	MCF-7 and MDA-MB-231 breast cancer cells	CLNCs and ULNCs	24 and 48 h	1) TQ + DTX in CLNCs and ULNCs significantly showed lower cell viability and higher cell inhibition than free DTX, free TQ, and DTX-LNCs. P < 0.05.
Zafar et al, 2020 [31]	India	DxTq-LNCs	DTX-LNCs, free DTX alone, free TQ alone	MCF-7 and MDA-MB-231 breast cancer cells	Long circulating sub-100 nm mPEG-DSPE- Vitamin E TPGS-LNCs	24 and 48 h	1) DxTq-LNCs significantly showed lower cell viability than free DTX alone and DTX-LNCs. P < 0.05. 2) TQ lowers the effective concentration (IC₅₀) of DTX in LNCs. 3) DxTq-LNCs showed 0.53-0.79 (synergistic effect).
El-Far et al, 2021 [32]	Egypt	TQ + DOX	DOX alone, untreated cell	MCF-7 breast cancer cell	-	5 days after DOX treatment and 24 h after TQ treatment	1) TQ + DOX and TQ alone significantly increased apoptosis compared to untreated cell compared to untreated cells (P < 0.001 and P < 0.05, respectively). 2) TQ significantly increased the Bax/Bcl2 ratio and caspase-3 activity compared to DOX alone and untreated cells. P < 0.01.
Zheng et al, 2022 [33]	China	TQ + 5-FU	5-FU alone	BT-549 and MDA-MD-231 breast cancer cells	-	24 and 48 h	1) 5-FU + TQ significantly showed lower cell viability, higher cell inhibition, and increased apoptosis than 5-FU or TQ alone. P < 0.01. 2) 5-FU + TQ was more effective in prolonging the S phase of the cell cycle than other treatments.
Anandan et al, 2023 [34]	India	TQ + PTX	PTX alone, TQ alone	MCF-7 breast cancer cells	-	24 h	1) TQ + PTX significantly showed lower cell viability than PTX and TQ alone. P < 0.05. 2) TQ lowers the effective concentration of PTX.
Bawadud et al, 2023 [35]	Saudi Arabia	NE-DTX + TQ, free DTX + TQ	Free DTX alone, free TQ alone, drug-free NEs	Human ductal carcinoma cells T47D	NEs	48 h	1) NE-DTX + TQ had a lower IC₅₀ than free DTX alone and other treatments. 2) Free TQ + DTX showed 7.9 (antagonistic effect), while NE-DTX + TQ showed 0.75 (synergistic effect). 3) NE-DTX + TQ significantly increased apoptosis (P < 0.0001) and reduced CD44⁺/CD24^- (P < 0.0002) compared to free DTX alone. 4) NE-DTX + TQ had the highest rate of autophagic vacuole formation compared to other treatments. P < 0.0001.
Loo et al, 2023 [36]	Malaysia	TQ + GCB, in LLCNs, free TQ + GCB	Free TQ alone, free GCB alone, drug-free LLCNs, untreated cell	MCF10A nonmalignant breast epithelial and MCF-7 breast cancer cell	LLCNs	24, 48, and 72 h	1) TQ + GCB in LLCNs had a lower IC₅₀ compared to drug-free LLCNs. 2) Free TQ + GCB and TQ + GCB in LLCNs significantly showed the lowest cell viability compared to other treatments. P < 0.05. 3) TQ + GCB in LLCNs showed 0.87 (synergistic effect), while free TQ + GCB showed 0.92 to 1.40 (additive to antagonistic effect).
Mousavinasab et al, 2023 [37]	Iran	TQ + Cis	TQ alone, Cis alone	MCF-7 breast cancer cell	-	24, 48, and 72 h	1) TQ + Cis significantly showed lower cell viability compared to Cis alone. P < 0.05. 2) TQ lowers the effective concentration of Cis. 3) TQ + Cis significantly induced the highest caspase-9, caspase-3, PPAR, p53, Bax, and the lowest Bcl-2 compared to other treatments. P < 0.00001.

Table 1. Characteristics and Main Outcomes of In Vitro Studies Included

Author (years)

Country

Intervention group

Control group

Cellular type

Carrier

Duration

Outcomes

ACNP: aragonite calcium carbonate (CaCO₃) nanoparticle; Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2; B-NE: borage nanoemulsion; Cis: cisplatin; CLNCs: chitosan-coated lipid nanocapsule; Cyclo: cyclophosphamide; DOX: doxorubicin; DTX: docetaxel; GCB: gemcitabine; 5-FU: 5-fluorouracil; LLCNs: lyotropic liquid crystalline nanoassemblies; LNCs: lipid nanocapsules; NE: nanoemulsion; p-5473-Akt: phosphorylated Akt; PLGA: poly lactic-co-glycolic acid; PPAR: peroxisome proliferator-activated receptor; PTEN: phosphatase and TENsin homolog; PTX: paclitaxel; TQ: thymoquinone; ULNCs: drug-loaded uncoated lipid nanocapsule.

Soni et al, 2015 [28]

India

TQ + PTX

PTX alone

MCF-7 breast cancer cell

PLGA nanoparticle

24 h

1) TQ + PTX significantly showed lower cell viability compared to PTX alone. P < 0.001.
2) TQ lowers the effective concentration (IC₅₀) of PTX.
3) TQ + PTX showed 0.688 (synergistic effect).

Sakalar et al, 2016 [29]

Turkey

TQ + PTX

PTX alone, untreated embryonic fibroblasts cell (PMEFCFL-P1)

4T1 (ATCC CRL-2539) breast cancer cells

48 h

1) TQ + PTX significantly showed lower cell viability compared to PTX alone. P < 0.0001.
2) Lower dose TQ showed higher apoptosis compared to untreated cells. P < 0.05.
3) TQ modulated apoptosis-related genes, cytokine, and p53 signaling pathway genes.

Bashmail et al, 2018 [40]

Saudi Arabia

TQ + GCB

GCB alone, untreated cell

MCF-7 and T47D breast cancer cells

24, 48, and 72 h

1) TQ lowers the effective concentration (IC₅₀) of GCB.
2) TQ + GCB showed 0.15 (strong synergistic effect).
3) TQ + GCB showed significantly higher apoptosis, increased cell death in the pre-G phase, and cell cycle arrest at S phase compared to GCB alone. P < 0.05.
4) TQ + GCB significantly increased autophagic cell death compared to control untreated cells. P < 0.05.

Zidan et al, 2018 [42]

Egypt

DOX + TQ + F2 gel, DOX + F2 gel, TQ + F2 gel

Free DOX, free TQ, untreated cell

MCF-7 breast cancer cells

F2 gel nanofibers

24 h

1) DOX + TQ + F2 gel showed lower cell viability compared to other treatments. P < 0.05.
2) DOX + TQ + F2 gel induced the highest apoptosis, 88.6%, compared to the control group.

Ibiyeye et al, 2019 [43]

Malaysia

DOX + TQ-ACNP, DOX-ACNP, TQ-ACNP

Free DOX + TQ, free TQ, free TQ

MDA-MB-231 breast cancer cell, MCF10A normal breast cell, 3T3 normal fibroblast cell

ACNP

24, 48, and 72 h

1) TQ + DOX showed lower cell viability than other treatments. P < 0.05.
2) Free TQ + DOX showed 0.8 at 48 h (slight synergism), while TQ + DOX-ACNP showed 0.5 at 48 h (synergism).
3) Free TQ increased the late apoptosis in TQ + DOX compared to DOX alone.
4) DOX + TQ-ACNP has the highest percentage in SubG0 (dead cells) and S phase at 48 h.
5) TQ has been shown to significantly inhibit cancer cell invasion and migration in TQ + DOX compared to other treatments. P < 0.05.

Khan et al, 2019 [44]

Saudi Arabia

TQ + cyclo

Cyclo alone, untreated cell

SKBR-3 and MDA-MB-231 breast cancer cells

48 h

1) TQ enhanced the growth inhibition of Her2⁺ and Her2^- breast cancer cells in combination with cyclo, inhibited p-5473-Akt, increased the expression of its inhibitor PTEN, and decreased cyclin D1 compared to cyclo alone. P < 0.001.
2) TQ + cyclo increased cells to arrest in sub-G1 and G1 compared to untreated cells. P < 0.05.

Odeh et al, 2019 [45]

Jordan

TQ + DTX

DTX alone

MCF-7 breast cancer cell

72 h

1) TQ lowers the effective concentration (IC₅₀) of DTX.
2) TQ + DTX showed 0.552 - 0.803 (synergistic effect).

Alkhatib et al, 2020 [46]

Saudi Arabia

TQ + DTX + B-NE

Free TQ and DTX

MCF-7 and MDA-MB-231 breast cancer cells

B-NE

48 h

1) TQ + DTX in B-NE exhibited a lower IC₅₀ than free DTX and other treatments.
2) TQ + DTX in B-NE showed 0.6-0.9 (synergistic effect).
3) TQ + DTX in B-NE significantly showed higher apoptosis compared to DTX alone. P < 0.05.
4) TQ + DTX and free TQ or DTX significantly showed higher autophagy than untreated cell. P < 0.05.

Bashmail et al, 2020 [47]

Saudi Arabia

TQ + PTX

PTX alone, TQ alone

MCF-7 and T47D breast cancer cells

24 and 48 h

1) TQ did not enhance PTX potency but significantly eliminated tumor-associated resistant cell clones to PTX. P < 0.05.
2) TQ + PTX showed 1.6 - 4.6 (antagonistic effect).
3) TQ + PTX significantly increased apoptosis, pre-G phase population, autophagic cell death fluorescence, and reduced CD44⁺/CD24^- compared to PTX alone. P < 0.05.

Bawadud et al, 2020 [48]

Saudi Arabia

NE-DTX + TQ, free DTX + TQ

Free DTX alone, free TQ alone, drug-free NEs

MCF-7 and MDA-MB-231 breast cancer cells

48 h

1) DTX + TQ-NE and free DTX alone significantly increased DNA fragmentation compared to free TQ alone and drug-free NEs. P < 0.05.
2) DTX + TQ-NE significantly reduced CD44⁺/CD24^-, SNAIL-1, and TWIST-1 compared to other treatments. P < 0.05.
3) DTX + TQ-NE caused significant arrest at the G2/M phase (P < 0.0001) and the S phase (P < 0.0021) compared to untreated cells and free TQ alone.

Zafar et al, 2020 [30]

India

DTX + TQ in ULNCs and CLNCs

Free DTX alone, free TQ alone, DTX-LNCs

MCF-7 and MDA-MB-231 breast cancer cells

CLNCs and ULNCs

24 and 48 h

1) TQ + DTX in CLNCs and ULNCs significantly showed lower cell viability and higher cell inhibition than free DTX, free TQ, and DTX-LNCs. P < 0.05.

Zafar et al, 2020 [31]

India

DxTq-LNCs

DTX-LNCs, free DTX alone, free TQ alone

MCF-7 and MDA-MB-231 breast cancer cells

Long circulating sub-100 nm mPEG-DSPE- Vitamin E TPGS-LNCs

24 and 48 h

1) DxTq-LNCs significantly showed lower cell viability than free DTX alone and DTX-LNCs. P < 0.05.
2) TQ lowers the effective concentration (IC₅₀) of DTX in LNCs.
3) DxTq-LNCs showed 0.53-0.79 (synergistic effect).

El-Far et al, 2021 [32]

Egypt

TQ + DOX

DOX alone, untreated cell

MCF-7 breast cancer cell

5 days after DOX treatment and 24 h after TQ treatment

1) TQ + DOX and TQ alone significantly increased apoptosis compared to untreated cell compared to untreated cells (P < 0.001 and P < 0.05, respectively).
2) TQ significantly increased the Bax/Bcl2 ratio and caspase-3 activity compared to DOX alone and untreated cells. P < 0.01.

Zheng et al, 2022 [33]

China

TQ + 5-FU

5-FU alone

BT-549 and MDA-MD-231 breast cancer cells

24 and 48 h

1) 5-FU + TQ significantly showed lower cell viability, higher cell inhibition, and increased apoptosis than 5-FU or TQ alone. P < 0.01.
2) 5-FU + TQ was more effective in prolonging the S phase of the cell cycle than other treatments.

Anandan et al, 2023 [34]

India

TQ + PTX

PTX alone, TQ alone

MCF-7 breast cancer cells

24 h

1) TQ + PTX significantly showed lower cell viability than PTX and TQ alone. P < 0.05.
2) TQ lowers the effective concentration of PTX.

Bawadud et al, 2023 [35]

Saudi Arabia

NE-DTX + TQ, free DTX + TQ

Free DTX alone, free TQ alone, drug-free NEs

Human ductal carcinoma cells T47D

NEs

48 h

1) NE-DTX + TQ had a lower IC₅₀ than free DTX alone and other treatments.
2) Free TQ + DTX showed 7.9 (antagonistic effect), while NE-DTX + TQ showed 0.75 (synergistic effect).
3) NE-DTX + TQ significantly increased apoptosis (P < 0.0001) and reduced CD44⁺/CD24^- (P < 0.0002) compared to free DTX alone.
4) NE-DTX + TQ had the highest rate of autophagic vacuole formation compared to other treatments. P < 0.0001.

Loo et al, 2023 [36]

Malaysia

TQ + GCB, in LLCNs, free TQ + GCB

Free TQ alone, free GCB alone, drug-free LLCNs, untreated cell

MCF10A nonmalignant breast epithelial and MCF-7 breast cancer cell

LLCNs

24, 48, and 72 h

1) TQ + GCB in LLCNs had a lower IC₅₀ compared to drug-free LLCNs.
2) Free TQ + GCB and TQ + GCB in LLCNs significantly showed the lowest cell viability compared to other treatments. P < 0.05.
3) TQ + GCB in LLCNs showed 0.87 (synergistic effect), while free TQ + GCB showed 0.92 to 1.40 (additive to antagonistic effect).

Mousavinasab et al, 2023 [37]

Iran

TQ + Cis

TQ alone, Cis alone

MCF-7 breast cancer cell

24, 48, and 72 h

1) TQ + Cis significantly showed lower cell viability compared to Cis alone. P < 0.05.
2) TQ lowers the effective concentration of Cis.
3) TQ + Cis significantly induced the highest caspase-9, caspase-3, PPAR, p53, Bax, and the lowest Bcl-2 compared to other treatments. P < 0.00001.

**Table 2.** Characteristics and Main Outcomes of *In Vivo* Studies Included
Author (years)	Country	Sample	Route	Intervention/dose	Control group	Animal (age)	Weight (g)	Duration	Outcomes
ALT: alanine transaminase; AST: aspartate transaminase; BUN: blood urea nitrogen; CK-MB: creatine kinase-MB; CLNCs: chitosan-coated lipid nanocapsule; DOX: doxorubicin; DTX: docetaxel; ECis: early cisplatin; GCB: gemcitabine; GSH: glutathione; LCis: late cisplatin; LDH: lactate dehydrogenase; MDA: malondialdehyde; NS: normal saline; PBS: phosphate buffer saline; PTX: paclitaxel; RBC: red blood cell; SOD: superoxide dismutase; TQ: thymoquinone; WBC: white blood cell.
Şakalar et al, 2016 [29]	Turkey	Total 20 (control = 6, low dose TQ = 6, high dose TQ = 8)	Intraperitoneal injection	0.64 mg/kg TQ + 1.25 mg/kg PTX, 2.4 mg/kg TQ + 1.25 mg/kg PTX	PTX alone, DMSO and/or 50% ethanol in ddH₂O	Female balb/c mice (8 - 12 weeks)	26 - 28	8 days (low dose group), 11 days (high dose group)	1) TQ significantly decreased tumor growth (weight) in PTX + TQ compared to PTX alone. P < 0.007 in lower dose and P < 0.001 in higher dose.
El-Ashmawy et al, 2017 [38]	Egypt	Ten/group (normal control, tumor control, F2 gel, free DOX, DOX + F2 gel, free TQ, TQ + F2 gel, and DOX + TQ + F2 gel)	Subcutaneous injection	DOX + F2 gel (200 µL with 100 µg of DOX), TQ + F2 gel (200 µL with 3 mg of TQ), and DOX + TQ + F2 gel (300 µL with 100 µg of DOX and 3 mg of TQ), free DOX (5 mg/kg), free TQ (3 mg/mouse)	Normal control, tumor control, F2 gel (100 µL/mouse)	Female albino mice (6 - 8 weeks)	18 - 22	28 days	1) TQ significantly decreased tumor growth (volume) and increased inhibition rate in DOX + TQ and TQ alone compared to DOX alone. P < 0.05. 2) DOX + TQ + F2 gel decreased Bcl-2 and P53 expression compared to other treatments. P < 0.05. 3) TQ reduces the side effects of DOX on the heart as evidenced by decreased cardiac markers when combined with DOX. P < 0.05.
Gomaa et al, 2018 [39]	Egypt	Five/group (PB, gold nanoparticles (AuNPs), AuNPs/TQ conjugate, AuNPs/TQ + Cisplatin 10, and AuNPs/TQ + Cisplatin 40)	Intraperitoneal injection	AuNPs (21.4 µg/mouse) (AuNPs group), AuNPs/TQ conjugate (1 mg/mouse) (AuNPs/TQ group), AuNPs/TQ (1 mg/mouse) plus cisplatin (10 µg/mouse) (AuNPs/TQ + Cis10 group) or AuNPs/TQ (1 mg/mouse) plus cisplatin (40 µg/mouse) (AuNPs/TQ + Cis40 group)	PBS	Female Swiss albino mice (6 - 8 weeks)	25 ± 2	6 days	1) TQ significantly decreased tumor growth (weight) and increased CD4⁺, CD8⁺, granulocytic, and monocytic cell populations in AuNPs/TQ + Cis compared to untreated cells. P < 0.05.
Mosalam et al, 2020 [41]	Egypt	Ten/group (ECis, ECis + ETQ, ECis + ETQ + EPTX, LCis, LCis + LTQ, and LCis + LTQ + LPTX, tumor control)	Cis and TQ was given intraperitoneal injection, PTX was given subcutaneous injection	7.5 mg/kg Cis (on 12th and 18th days for early groups, while on 19th and 25th days for late groups), 20 mg/kg TQ, 15 mg/kg PTX (TQ and PTX injected daily for 10 days)	ECis alone, LCis alone, tumor control PEG 200 µL/mouse/day	Female albino mice (6 - 8 weeks)	20 - 22	28 days	1) TQ significantly decreased tumor growth (weight), increased inhibition rate, CD4⁺, CD8⁺, and apoptosis rate in ECis + ETQ + EPTX and LCis + LTQ + LPTX compared to ECis or LCis alone and untreated cells. P < 0.05.
Zafar et al, 2020 [30]	India	Ten/group (control and TQ groups)	Direct contact	3.3 µM DTX + 6.6 µM TQ in CLNCs carrier	DTX alone, TQ alone, NS	Chick Embryo Chorioallantoic Membran (CAM) (9 days)	-	1 and 2 days	1) TQ increased vascular inhibition (antiangiogenic effect) in TQ + DTX-CLNCs compared to DTX alone and untreated cells.
Zafar et al, 2020 [31]	India	Total 9 (NS = 3, 2 DTX = 3, DTX + TQ co-encapsulated lipid nanocapsules (DxTq-LNCs) = 3)	Intravenous tail vein injection	DTX + TQ - LNCs (equivalent to 0.3 mg DTX and 0.6 mg TQ) containing 2 mg/kg DTX	DTX alone, NS	Female balb/c mice	20 - 25	14 days	1) TQ significantly increased inhibition rate (volume) in DTX + TQ - LNCs compared to DTX alone. P < 0.05. 2) TQ significantly reduces liver, kidney, and blood toxicity of DTX as indicated by elevated SOD and GSH levels, reduced MDA, AST, and ALT, along with decreased BUN and creatinine, and increased WBC and RBC counts in DTX + TQ - LNCs. P < 0.05.

Table 2. Characteristics and Main Outcomes of In Vivo Studies Included

Author (years)

Country

Sample

Route

Intervention/dose

Control group

Animal (age)

Weight (g)

Duration

Outcomes

ALT: alanine transaminase; AST: aspartate transaminase; BUN: blood urea nitrogen; CK-MB: creatine kinase-MB; CLNCs: chitosan-coated lipid nanocapsule; DOX: doxorubicin; DTX: docetaxel; ECis: early cisplatin; GCB: gemcitabine; GSH: glutathione; LCis: late cisplatin; LDH: lactate dehydrogenase; MDA: malondialdehyde; NS: normal saline; PBS: phosphate buffer saline; PTX: paclitaxel; RBC: red blood cell; SOD: superoxide dismutase; TQ: thymoquinone; WBC: white blood cell.

Şakalar et al, 2016 [29]

Turkey

Total 20 (control = 6, low dose TQ = 6, high dose TQ = 8)

Intraperitoneal injection

0.64 mg/kg TQ + 1.25 mg/kg PTX, 2.4 mg/kg TQ + 1.25 mg/kg PTX

PTX alone, DMSO and/or 50% ethanol in ddH₂O

Female balb/c mice (8 - 12 weeks)

26 - 28

8 days (low dose group), 11 days (high dose group)

1) TQ significantly decreased tumor growth (weight) in PTX + TQ compared to PTX alone. P < 0.007 in lower dose and P < 0.001 in higher dose.

El-Ashmawy et al, 2017 [38]

Egypt

Ten/group (normal control, tumor control, F2 gel, free DOX, DOX + F2 gel, free TQ, TQ + F2 gel, and DOX + TQ + F2 gel)

Subcutaneous injection

DOX + F2 gel (200 µL with 100 µg of DOX), TQ + F2 gel (200 µL with 3 mg of TQ), and DOX + TQ + F2 gel (300 µL with 100 µg of DOX and 3 mg of TQ), free DOX (5 mg/kg), free TQ (3 mg/mouse)

Normal control, tumor control, F2 gel (100 µL/mouse)

Female albino mice (6 - 8 weeks)

18 - 22

28 days

1) TQ significantly decreased tumor growth (volume) and increased inhibition rate in DOX + TQ and TQ alone compared to DOX alone. P < 0.05.
2) DOX + TQ + F2 gel decreased Bcl-2 and P53 expression compared to other treatments. P < 0.05.
3) TQ reduces the side effects of DOX on the heart as evidenced by decreased cardiac markers when combined with DOX. P < 0.05.

Gomaa et al, 2018 [39]

Egypt

Five/group (PB, gold nanoparticles (AuNPs), AuNPs/TQ conjugate, AuNPs/TQ + Cisplatin 10, and AuNPs/TQ + Cisplatin 40)

Intraperitoneal injection

AuNPs (21.4 µg/mouse) (AuNPs group), AuNPs/TQ conjugate (1 mg/mouse) (AuNPs/TQ group), AuNPs/TQ (1 mg/mouse) plus cisplatin (10 µg/mouse) (AuNPs/TQ + Cis10 group) or AuNPs/TQ (1 mg/mouse) plus cisplatin (40 µg/mouse) (AuNPs/TQ + Cis40 group)

PBS

Female Swiss albino mice (6 - 8 weeks)

25 ± 2

6 days

1) TQ significantly decreased tumor growth (weight) and increased CD4⁺, CD8⁺, granulocytic, and monocytic cell populations in AuNPs/TQ + Cis compared to untreated cells. P < 0.05.

Mosalam et al, 2020 [41]

Egypt

Ten/group (ECis, ECis + ETQ, ECis + ETQ + EPTX, LCis, LCis + LTQ, and LCis + LTQ + LPTX, tumor control)

Cis and TQ was given intraperitoneal injection, PTX was given subcutaneous injection

7.5 mg/kg Cis (on 12th and 18th days for early groups, while on 19th and 25th days for late groups), 20 mg/kg TQ, 15 mg/kg PTX (TQ and PTX injected daily for 10 days)

ECis alone, LCis alone, tumor control PEG 200 µL/mouse/day

Female albino mice (6 - 8 weeks)

20 - 22

28 days

1) TQ significantly decreased tumor growth (weight), increased inhibition rate, CD4⁺, CD8⁺, and apoptosis rate in ECis + ETQ + EPTX and LCis + LTQ + LPTX compared to ECis or LCis alone and untreated cells. P < 0.05.

Zafar et al, 2020 [30]

India

Ten/group (control and TQ groups)

Direct contact

3.3 µM DTX + 6.6 µM TQ in CLNCs carrier

DTX alone, TQ alone, NS

Chick Embryo Chorioallantoic Membran (CAM) (9 days)

1 and 2 days

1) TQ increased vascular inhibition (antiangiogenic effect) in TQ + DTX-CLNCs compared to DTX alone and untreated cells.

Zafar et al, 2020 [31]

India

Total 9 (NS = 3, 2 DTX = 3, DTX + TQ co-encapsulated lipid nanocapsules (DxTq-LNCs) = 3)

Intravenous tail vein injection

DTX + TQ - LNCs (equivalent to 0.3 mg DTX and 0.6 mg TQ) containing 2 mg/kg DTX

DTX alone, NS

Female balb/c mice

20 - 25

14 days

1) TQ significantly increased inhibition rate (volume) in DTX + TQ - LNCs compared to DTX alone. P < 0.05.
2) TQ significantly reduces liver, kidney, and blood toxicity of DTX as indicated by elevated SOD and GSH levels, reduced MDA, AST, and ALT, along with decreased BUN and creatinine, and increased WBC and RBC counts in DTX + TQ - LNCs. P < 0.05.

**Table 3.** Summary of Beneficial and Adverse Effects of TQ-Chemotherapy Combination in Clinical Practice
Benefit	Adverse effect
TQ itself has a direct anticancer effect and increases the immune response [29, 39-41].	Potential drug interactions and toxicities depend on the concentration of TQ, drug carrier, and combination chosen [35, 36, 47, 54].
Enhance the efficacy of various chemotherapeutic agents [28, 31, 34, 37, 40, 45].	Possible variability in patient response [88].
Reducing chemotherapy-induced toxicity [31, 38, 39].
Improve cost-effective adjunct chemotherapy by reducing the required doses of conventional treatments [28, 31, 34-37, 40, 43, 45, 46].

Table 3. Summary of Beneficial and Adverse Effects of TQ-Chemotherapy Combination in Clinical Practice

Benefit

Adverse effect

TQ itself has a direct anticancer effect and increases the immune response [29, 39-41].

Potential drug interactions and toxicities depend on the concentration of TQ, drug carrier, and combination chosen [35, 36, 47, 54].

Enhance the efficacy of various chemotherapeutic agents [28, 31, 34, 37, 40, 45].

Possible variability in patient response [88].

Reducing chemotherapy-induced toxicity [31, 38, 39].

Improve cost-effective adjunct chemotherapy by reducing the required doses of conventional treatments [28, 31, 34-37, 40, 43, 45, 46].