Effects of Berberine on Liver Cancer

Abstract

Liver cancer, otherwise known as hepatocellular carcinoma, is a chronic disease condition with an excessive deposition and growth of malignant cells in the body. The high incidence and prevalence rates of liver cancer continue to be problems, as well as its poor prognosis and therapeutic limitations involving severe drug adverse reactions linked to the use of synthetic chemotherapeutic compounds. Continuous experimental studies, as well as utilization of pure herbal-based compounds, are essential towards finding more potent cures for liver cancer. Natural bioactive compounds, particularly alkaloids (eg, berberine), have been shown to be highly beneficial in the treatment of various diseases. Berberine (BBR), an isoquinoline alkaloid, is obtained from stem, bark, roots, rhizomes, and leaves of several medicinal plants, including Berberis species. It is commonly synthesized from the benzyltetrahydroisoquinoline system with the incorporation of an additional carbon atom as a bridge. The multiple attributes of BBR involving effective inhibitory and cytotoxic actions against the proliferation of cancer cells have been demonstrated. The use of BBR in experimental studies (in vivo and in vitro) for over a decade for liver cancer treatment has proven to be highly effective, safe, and potent. Until now, the poor solubility of BBR remains one of the contributing factors leading to its minimal clinical bioavailability. Therefore, BBR could serve as a prospective drug candidate in the future towards drug formulation for liver cancer treatment. The relevant information regarding this review was obtained electronically through the use of databases such as PubMed, Google Scholar, Springer, Hindawi, Embase, Web of Science, and China National Knowledge Infrastructure. All the aforementioned databases were searched from 1981 to 2020. This literature represents an update of previous review papers discussing the various positive pharmacological and mechanistic effects (oxidative stress regulation, inflammation reduction, apoptosis activation, overcoming drug resistance, and metastasis inhibition) of BBR for liver cancer treatment, which would be of great significance to drug development and clinical research.

Introduction

Liver cancer is termed a chronic disease with an excessive or uncontrolled deposition and proliferation of malignant cells in the body.^1,2 It is normally accompanied by a manifestation of symptoms like swelling of the abdomen, weakness, weight loss, yellowish skin, and easy bruising and pains in the right side below the rib cage. The occurrence of liver cancer is associated with certain causative agents such as iron overload, obesity, hepatitis-B virus, diabetes, smoking, alcohol-related cirrhosis, liver flukes, hepatitis-C virus, nonalcoholic fatty liver disease (NAFLD), aflatoxin exposure, immunosuppression, and lupus (systemic lupus erythematosus).^3–5 The prevalence of liver cancer is still rapidly on the rise worldwide, with a minimal survival rate.^6,7 This, combined with poor prognosis and therapeutic shortcomings or limitations involving chronic drug adverse reactions to the synthetic chemotherapeutic components applied in drug formulation are causes of concern. The treatments often delay, but can potentiate the degree of disease progression. More experimental studies, as well as utilization of pure herbal-based compounds, are essential towards finding more potent curative agents to treat liver cancer. ⁸

Natural bioactive compounds, particularly alkaloids (eg, Berberine; BBR), have been shown to be highly beneficial in the treatment of various diseases.^9–14 BBR is commonly synthesized from the benzyltetrahydroisoquinoline system with the incorporation of an additional carbon atom as a bridge. Current evidence indicates the multiple attributes of BBR involving effective inhibitory and cytotoxic actions (oxidative stress regulation, inflammation reduction, apoptosis, and autophagy activation) against the proliferation of cancer cells.^15–21 The use of BBR in experimental studies (in vivo and in vitro) for over a decade against liver cancer has proven to be highly effective, safe, and potent. However, until now, the poor solubility of BBR remains one of the contributing factors leading to its minimal clinical bioavailability. Therefore, BBR could serve as a prospective drug candidate for drug formulation against liver cancer. This work represents an update of previous review papers discussing the various positive pharmacological and mechanistic effects (oxidative stress regulation, inflammation reduction, apoptosis activation, overcoming drug resistance, and metastasis inhibition) of BBR for liver cancer treatment.

Methodology

The information shown in this review was obtained electronically through the use of databases such as PubMed, Google Scholar, Springer, Hindawi, Embase, Web of Science, and China National Knowledge Infrastructure by using keywords such as “Berberine,” “Berberine and Cancer,” “Berberine and Tumor,” “Berberine and Carcinoma,” “Berberine against liver cancer,” “Berberine against hepatocellular carcinoma,” Hepatocellular carcinoma,” Liver cancer,” and “Berberine effects on liver cancer.’’ All the aforementioned databases were searched from 1981 to 2020.

Botanical Sources, Pharmacological Activities, and Metabolism of BBR

The isoquinoline alkaloid, BBR (Figure 1) or 5,6-dihydro-9,10-dimethoxybenzo[g]-1-3-benzodioxolo[5,6-a] quinolizinium derivative, is a constituent of many different medicinal plants such as Berberis species (B. aquifolium, B. vulgaris, B. heterophylla, B. darwinii, B. petiolaris, B. beaniana, and B. aristata), and others (eg, Hydratis canadensis, Phellodendron chinense, Coptidis sp., Argemone mexicana, Camellia japonica, Tinospora cordifolia, Coscinium fenestratum, Xanthorhiza simplicissima, Callosobruchus chinensis, and Phellodendron amurense).^22–27 Table 1 lists the BBR containing species (family, botanical names) and their applications. Owing to their yellowish color, Berberis species are commonly utilized in dyeing leather, wood, and wool. BBR also has a wide-ranging history in Asia, being known for its low or poor lipid solubility alongside its remarkable biochemical and pharmacological activities.^50–53 Traditionally, BBR, particularly BBR sulfate and hydrochloride, is extensively applied in traditional Chinese and Ayurvedic medicine.^54,55

Figure 1.

Chemical structure of berberine.

Table 1.

Berberine Containing Species and Their Applications.

No	Family	Botanical name	Application	References
1	Menispermaceae	Tinospora sinensis (Lour.) Merr {ex Tinospora cordifolia (Willd.) Miers}	Utilized as anti-spasmodic, anti-stress, anti-allergic, anti-malarial, tonic, anti-diabetic, anti-arthritic, anti-inflammatory and anti-periodic agents.	28,29
2	Rutaceae	Zanthoxylum monophyllum Tul.	Treats dark vomitus and ophthalmic inflammation	30
		Phellodendron amurense Rupr.	Used in treating cancer, diarrhea, abdominal pain, gastroenteritis and possesses immunostimulating and anti-inflammatory activities	31
		v (ex Phellodendron wilsonii Hayata & Kaneh.). v Phellodendron chinense var. glabriusculum C. K. Schneid. v Phellodendron chinense C. K. Schneid.	Treats night sweats, vaginal infections, conjunctivitis, skin diseases, jaundice, meningitis, boils, dysentery, acute urinary tract infection, and diarrhea.Serves as vasodilator, aphrodisiac, and expectorantUsed as anti-rheumatic and anti-malarial agents, and detoxicant for hot damps related conditions to the kidney.	32
3	Annonaceae	Annickia polycarpa (DC.) Setten & Maas ex I.M.Turner (ex-Enantia polycarpa (DC.) Engl. & Diels)	Treats intestinal problems, jaundice, feverish conditions associated with malaria, ophthalmic problems, skin infections, sores, cuts, and improves wound healing.	32, 33
		Annickia chlorantha (Oliv.) Setten & Maas (ex Enantia chlorantha Oliv.)	Treats typhoid and yellow fever, boils, diabetes, vomiting, hypertension, urinary tract infections, malaria, cough, sexual asthenia, intestinal worms, tuberculosis, wounds, aches, syphilis, sores, conjunctivitis, jaundice, intestinal spasm, rheumatism, rickettsia fever, prostate cancer, hepatitis A, B, C, and D, dysentery, leishmaniosis, fatigue, sleeping sickness and improves conception.	32, 34–36
		Xylopia polycarpa (DC.) Oliv.	Promotes conception and utilized in treating malaria, leprosy, sleeping disorders, ulcer, diarrhea, ophthalmic diseases, rheumatism, fever, gall bladder, and stomach problems.	32
		Annickia pilosa (Exell) Setten & Maas (ex Enantia pilosa Exell)	Applied in the treatment of cuts	32
		Rollinia mucosa (Jacq.) Baill.	Applied in the treatment of tumors	32
4	Papaveraceae	Papaver hybridum L.	Serve as diuretic, sedative, anti-infective and anti-tussive agentsApplied in the treatment of dermatological diseases	32
		Glaucium corniculatum (L.) Rud. subsp. corniculatum	Treats central nervous system disturbancesUsed as anti-tussive, laxative, and sedative agents	37, 38
		Papaver rhoeas L. var. chelidonioides	Treats nervous digestive disorders, jaundice, fever, bronchial coughs, insomnia, painful conditions.Applied as sedative, emollient, narcotic, and anticancer agents.	32
		Papaver dubium L., Papaver dubium var. lecoquii	Serves as expectorant, ophthalmic, sudorific, and diuretic agents	32
		Macleaya microcarpa (Maxim.) Fedde	Treats inflammation and some skin disorders	39
		Macleaya cordata (Willd.) R.Br.	Treats ringworm, and insect bites Utilized as diuretic, analgesic, and anti-edemic agents	32
		Eschscholzia californica Cham.	Applied in the treatment of insomnia, pain, urinary incontinence, anxiety, spasms, nervous tension, and toothache Promotes perspiration and reduces the flow of milk in lactating mothers.	32
		Bocconia frutescens L.	Treats respiratory tract infections (tuberculosis and bronchitis), and skin conditions (ulcers)	32
		Argemone platyceras L.	Used in treating pneumonia, asthma, bronchitis, and cough	32
		Argemone albiflora Hornem (ex-Argemone alba F. Lestib.)	Serves as emetic, purgative and diuretic agents Applied in the treatment of wounds, colds, jaundice, and skin ailments	32
		Argemone mexicana L.	Utilized as antidote for snake poisoning, sedative, anti-spasmodic, analgesic, laxative, emetic agents Treats cataract, warts, chronic skin disease, cough, itches, and cold sores	32
		Corydalis solida subsp. brachylova Corydalis solida subsp. Slivenensis (Velen.) Hayek (ex-Corydalis slivenensis Velen.); Corydalis solida subsp. tauri cola;Corydalis turtschaninovii Besser (ex-Corydalis ternata (Nakai) Nakai)	Treats traumatic injury, lumbago, dysmenorrhea, hallucinogenic, rheumatism, cardiac arrhythmia disease, memory dysfunction, duodenal and gastric ulcer; serves as antibacterial, CNS stimulant, antispasmodics, and sedative for insomnia; lowers blood pressure, and calms the nerves. Treat gastric and duodenal ulcers, memory dysfunction, dysmenorrhea, rheumatism, and cardiac arrhythmia disease.	40, 41
		Chelidonium majus L.	Treat warts, whooping cough, ophthalmic related conditions, bronchitis, asthma, gallstones, stomach ulcer, ringworm, jaundice, and gallbladder painsServes as sedative, antispasmodic, intestinal and muscle (bronchial tubes) relaxant, antispasmodic, diuretic, narcotic, purgative, anticancer, and analgesic agents.	32
5	Berberidaceae	Sinopodophyllum hexandrum (Royle) T. S. Ying	Improves the circulation of blood and modulates menstruationTreats difficult labor, retention of dead fetus or placenta, and amenorrhea	42
		Caulophyllum thalictroides (L.) Michaux	Applied in the treatment of cramps, rheumatism, colic, hysteria, and menstrual cramps.Serves as uterine stimulant, antispasmodic, and inducer of menstruation.Promotes prompt delivery, and relieves the pain of childbirth	32
		Mahonia napaulensis DC.	Used in treating eyes-related inflammation, and dysentery.Utilized as demulcent and diuretics agents.	32
		Jeffersonia diphylla (L.) Pers.	Treats ulcers, sores, urinary problems, and diarrheaUtilized as emetic, antispasmodic, diuretic, and expectorant in coughs.	32
		Nandina domestica Thunb.	Used in treating fever in influenza, indigestion, acute bronchitis, muscles and traumatic injuries, tooth abscess, whooping cough, and pain in the bones.Serves as anti-rheumatic, astringent, and anti-tussive	32
		Mahonia fortunei (Lindl.) Fedde	Applied as anti-odontalgic, anticancer, and treating arthritic pain and testicular swelling or inflammation	43
		Berberis aquifolium Pursh	Utilized in the treatment of jaundice, hemorrhages, fungal infections, eczema, dysentery, cirrhosis, acne, herpes, hepatitis, sore womb following menstruation or childbirth, psoriasis, digestive problems, conjunctivitis, gall bladder diseases, constipation, stomach problems, skin conditions, and few forms of cancer.Stimulates bile flow, intestinal secretion, and promotes blood flow to the liver	44
		Berberis asiatica Roxb. ex DC.	Treats ear and eye diseases, rheumatism, hyperpigmentation, headache, diabetes mellitus, malarial fever, asthma, jaundice, toothache, wounds, pneumococcal infections, stomach disorders, inflammation, and ulcers	32
		Berberis actinacantha Mart.	Anti-pyretic	45
		Berberis pseudumbellata R. Parker	Treats ulcer, sore throat, jaundice, stomach problems, eye diseases, and intestinal disorders	46
		Berberis vulgaris L.	Serves as antiarrhythmic, sedative, and anticancer agentsUsed in treating fever, sore throat, internal injuries, and kidney stones	47
		Berberis microphylla G. Forst. (ex Berberis heterophylla Juss. ex Poir.)	Applied in the treatment of diarrhea, inflammation, and febrifuge	32
		Berberis thunbergii DC.	Anti-inflammatory	48
		Berberis oblonga (Regel) C. K. Schneid	Treats arthralgia, diarrhea, pyrexia, back pain, jaundice, eye diseases, stomach ache, and mouth-related wounds	32
		Berberis tinctoria Lesch.	Treats menorrhagia, rheumatism, skin disease, and diarrhea	32
		Berberis petiolaris Wall. ex G. Don	Treats jaundice, conjunctivitis, diarrhea, and malarial fever	49
		Berberis umbellata Wall. ex G. Don	Utilized in the treatment of skin problems, eye disorders, fever, nausea, and jaundice	32
		Berberis darwinii Hook.	Treats fever, stomach pains, colitis, and indigestion	32
		Berberis jaeschkeana C. K. Schneid.	Treats eye-related diseases	32
		Berberis lycium Royle	Used in the treatment of pimples, eye diseases, sore throat, cough, jaundice, diabetes, diarrhea, menorrhagia, bone fractures, piles, intestinal problems, backache, sun blindness, dysentery, earache, fracture, eye ache, stomach ache, boils, wound healing, and scabies	32
		Berberis empetrifolia Lam.	Applied in the treatment of mountain sickness	45
		Berberis libanotica Ehrenb. ex C. K. Schneid.	Treats arthritis, neuralgic diseases, rheumatic, and muscular pain	32
		Berberis integerrima Bunge.	Treats chest pain, diabetes, headaches, bone fractures, constipation, rheumatism, tuberculosis, wound, heart pain, kidney stones, and stomach aches	32
		Berberis leschenaultia Wall. ex Wight & Arn.	Treats cold, complications during post-natal period, and serves as antipyretic	32
		Berberis koreana Palib.	Serves as antipyreticUtilized in treating sore throats, gastroenteritis, and conjunctivitis	32
		Berberis chitria Buch.-Ham. ex-Lindl.	Treats ophthalmic conditions, skin disease, ulcers, jaundice, rheumatism, ulcers, enlarged liver, and spleen	32
		Berberis aristata DC.	Treats dysentery, osteoporosis, jaundice, urinary tract infections, eye condition, allergies, cholera, metabolic disorders, fever, diarrhea, malaria, skin related diseases, piles, and menorrhagia	32
		Berberis buxifolia Lam.	Used in treating infections	32
6	Ranunculaceae	Hydrastis canadensis L.	Treats disorders of the digestive system and mucous membranes, constipation, disorders affecting the ears, eyes, throat, nose, stomach, intestines, and vagina	32
		Coptis chinensis Franch.	Control of bacterial and viral infections, relax spasms, and lower feversTreats diabetes mellitus, diarrhea, acute enteritis, and dysentery, treat insomnia, leukemia, otitis media, conjunctivitis, skin problems, toothache, swollen gums, mouth and tongue ulcers	32
		Xanthorhiza simplicissima Marshall	Treats jaundice, mouth ulcers, digestive disorders, stomach ulcers, piles, and cold	32
		Coptis teeta Wall.	Applied in the treatment of pectoral diseases, ophthalmic conditions, spasms, dysentery, and fevers.Controls bacterial and viral infections, and stimulates circulation,	32
		Coptis japonica (Thunb.) Makino	Treats intestinal catarrh, conjunctivitis, dysentery, high fevers, enteritis, inflamed tongue, and mouth.Stimulates circulation, relaxes spasms, and controls bacterial and viral infections	32

Recent analytical studies have shown that BBR could serve varying purposes, such as anti-obesity, anti-microbial, spatial memory enhancement, anti-diabetic, anti-cancer, anti-atherosclerotic, anti-hypertensive, anti-hyperlipidemic, anti-protozoal, anti-inflammatory, neuroprotective, hepatoprotective, and anti-oxidative agents,^56–62 as well as being utilized clinically for the treatment of disease conditions involving polycystic ovary syndrome, NAFLD, CAD, and metabolic syndrome. Table 2 summarizes the numerous beneficial activities of BBR in animal models. The metabolism of BBR is considered mostly to take place in the liver via demethylation (phase I) and glucuronidation processes (phase II), and excreted in the bile. ⁸¹ Excessive consumption or administration of BBR in large doses (4-6 weeks) results in liver overload. Also, its usage in pregnancy may lead to miscarriage and contraction of the uterus, as well as neonatal jaundice. Other possible toxicological effects or adverse reactions include gastric troubles, headache, digestive problems, stomach upset, constipation, diarrhea, and flatulence.

Table 2.

In Vivo Beneficial Activities Associated With Berberine.

No	Condition	Details of assay (animal model)	Experimental effects	Ref.
1	Intestinal inflammation and gut microbiome	db/db mice	Diminishes body weight, food intake, blood glucose, intestinal inflammation, serum LPS, and HbA1c levels. Restores intestinal SCFA content and barrier structure. Promotes the number of SCFA-producing bacteria and reduces opportunistic pathogens.	63
2	Hypertension	Spontaneously hypertensive rats	Down-regulates the elevated levels of aldosterone, collagen IV, angiotensin II, collagen III, IL-23, IL-6, IL-17, albumin, osteopontin, and KIM-1.	64
3	Diabetic retinopathy	STZ induced diabetic retinopathy in male Sprague-Dawley mice	Hinders cell apoptosis, ganglion cell layer, oxidative stress, and deactivates NF-kB signaling pathway	65
4	Diabetic nephropathy	HFD and STZ induced diabetic nephropathy rats	Inhibits elevated levels of biochemical indicators, and effectively potentiates the abnormal expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), and phosphorylated Akt.	66
		STZ induced diabetic nephropathy rats	Inhibits renal injury, fasting blood glucose, ratio of kidney weight to body weight, 24-h urinary protein, serum creatinine, blood urine nitrogen, systemic and renal cortex inflammatory response, and TLR4/NF-kB pathway.	67
5	Diabetic encephalopathy	db/db and C57BL/6J-db/m mice	Enhances lipid metabolism, synapse and nerve-related protein expression (NGF, SYN, and PSD95), and protein expression of SIRT1. Downregulates fasting blood glucose, protein expression of inflammatory factors (NF-kB and TNF-α), and ER stress-associated proteins (IRE-1α, CHOP, PDI, PERK, and eIF-2α).	68
6	Axonopathy and diabetic encephalopathy	HFD and STZ induced diabetic rats	Decreases blood glucose, tau hyperphosphorylation, axonopathy, memory impairment, insulin level, insulin resistance, and restores PI3K/Akt/GSK3β signaling pathway	69
7	Diabetes	STZ induced diabetic albino Wistar mice	Significantly suppresses hepatic markers, lipid peroxidation markers (LOOH and TBARS), pro-inflammatory mediators (TNF-α, phospho-NF-kB p65, COX-2, and iNOS), and pro-apoptotic mediators (Bax and cytochrome c). Potentiates hexokinase, glucose-6-phosphate dehydrogenase, enzymatic antioxidant (SOD, CAT, and GPx), non-enzymatic antioxidants (GSH, vitamin E, and vitamin C), and anti-apoptotic protein (Bcl).	70,71
		Alloxan and HFD induced diabetic Wistar mice	Represses the fasting blood glucose level, serum content of LDL-c, TG, and TC. Improves HDL-c, NO, SOD, and GSH-px. Obstructs the increase of MDA and restored the damage to pancreas tissues.	72
		HFD and STZ induced diabetic mice	Down-regulates the levels of fasting blood glucose, LDL-c, total cholesterol, hypothalamic orexin-A, OX2R receptor, corticotropin-releasing hormone, pituitary and plasma adrenocorticotropic hormone, serum and urine corticosterone. Promotes the insulin sensitivity index, abnormalities of HDL-c, insulin resistance index, insulin levels, glucagon, mRNA, and protein expressions of GLUT4 in skeletal muscles	73
8	Alzheimer's disease and type-2 diabetes mellitus	STZ and Aβ25−35 induced Alzheimer’s diabetic mice	Attenuates memory deficits, ER stress, and the increased levels of triglyceride, total cholesterol, fasting blood glucose, and glycosylated serum protein. Restores the disordered arrangements of nerve cells and up-regulates TUNEL-positive cells.	74
9	Alzheimer's disease	APP/PS1 transgenic rats	Enhances learning and memory, and glutathione (GSH) activity. Reduces hyperphosphorylated tau protein, lipid peroxidation, and NF-kB activity.	75
		APP/PS1 transgenic rats	Inhibits the levels of Aβ, sAPP-β, BACE1, PS1, Pen-2, and Aph-1α. Promotes the levels of ADAM17, sAPPα, ADAM10, learning, and memory.	76
10	Anxiety	Male Wistar mice	Alleviates locomotor activity, relapse, and anxiety-related behaviors. Increases TLR4, Sirt1, and α-actin activation.	77
11	Ulcerative colitis	DSS-induced mice	Diminishes the expression of IL-12, IL-6, IFN-γ, IL-1, IL-1β, TNF-α, MPO, iNOS, MDA and TGF-β. Up-regulates the expression of IL-10, IL-4 and SIgA.	78
12	Myocardial infarction	Male wild-type (C57BL6) rats	Promotes miR-29b expressional level, angiogenesis, and heart functions. Reduces infarct size.	79
13	Myocardial hypertrophy	CAA induced myocardial hypertrophy mice	Decreases the expression of β myosin heavy chain, atrial natriuretic peptide, and myocardial infarction associated transcript. Halts up-regulation of mRNA expression and downregulation of beclin 1 and autophagy-related 5.	80

Potential Utilization of BBR for the Treatment of Liver Cancer

Over the past decade, diverse studies carried out by researchers regarding the medicinal effects of BBR in the treatment of liver cancer cells portrayed or indicated certain clinical outcomes through suppression of cellular proliferation, activation of apoptosis, halting the process of angiogenesis, and slowing down the onset of metastases. In this respect, we outline the prospective in vivo and in vitro actions of BBR in liver cancer (Table 3).

Table 3.

Mechanistic Actions of Berberine on Liver Cancer.

No	Condition	Assay model (animal/cell/tissues)	Experimental effects	Ref.
1	Liver cancer	SMMC-7721 and HepG2 cells	Up-regulates the expression levels of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, and reduces vascular endothelial growth factor and anti-apoptotic protein B-cell lymphoma 2.	82
		Huh7 and HepG2 cells	Down-regulates the expression of Nrf2 signaling-related protein (Nrf2, HO-1, and NQO-1). Potentiates the radiation-induced oxidative stress and apoptosis.	83
		Human hepatoma HepG2 cell	Potentiates the levels of nitric oxide and reactive oxygen species	84
		MHCC-97L cell	Diminishes cellular proliferation, invasiveness, and HIF-1α/VEGF signaling pathway. Decrease the transcription level of Id-1 via suppressing its promotor activity.	85
		Bel-7402 and SMMC-7721 cells	Decreases the expression of cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-kB), urokinase-type plasminogen activator, and matrix metalloproteinase-9 (MMP-9). Inactivates p38 and Erk1/2 signaling pathway.	86
		Human hepatocarcinoma (HepG2) cell	Increases the mRNA expression of FoxO1 and FoxO3a. Improves JNK phosphorylation, ROS generation, and lipid peroxidation. Reduces level of catalase, superoxide dismutase, and glutathione.	87
		H22, HepG2, and Bel-7404 cells	Reduces the protein expression levels of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2. Up-regulates the content ratio of arachidonic acid to prostaglandin E2.	88
		HepG2, Bel-7402, and SMMC-7721	Hampers the expression of cyclin D1, cyclin E, and cdc 2 and reduces the phosphorylation of Akt, mTOR, and ERK.	89
		BALB/c nude mice, Hep3B, and BEL-7404 cells	Suppresses the growth of HCC cells in vitro. Inhibits the glutamine uptake by diminishing SLC1A5. Decreases the proliferation of tumor xenografts, and the expression of SLC1A5 and c-Myc in-vivo.	90
		Hep3B, HepG2, HEK293, and Huh7 cells	Modulates β-catechin pathway independent of AMPK. Antagonizes β-catechin pathway via targeting Cap-dependent translation. Regulates β-catechin expression at the level of translation. Inhibits mTOR pathway and Cap-dependent translation. Activates HCC cell apoptosis via antagonizing Cap-dependent translation and β-catechin axis.	91
		Human hepatocarcinoma (HepG2) cell	Triggers the activation of caspase-8 and caspase-3, release of cytochrome c and PARP (poly ADP-ribose polymerase) cleavage, and down-regulates expression of Bid and anti-apoptosis factor Bcl-XL	92
		HepG2, SMMC-7721, and Bel-7402 cell	Significantly elevates phosphorylated AMP-activated protein kinase, phosphorylated Akt level, and Bax/Bcl-2 ratio in a dose-dependent manner.	93
		Human hepatoma WRL68 cells	Improves the expression of Bax. Regulates the protein expression of Bcl-2 associated with caspase -3/7 activities.	94
		Human hepatoma HepG2 cell	Diminishes the expressions of Bcl-2 protein and pro-caspase-3, and promotes Bax protein.	95
		Human hepatocarcinoma (HepG2) cell	Down-regulates the NF-kB p65 level.	96
		Human hepatocarcinoma (HepG2) cell	Reverses the adhesion and migration of HepG2 cells via suppressing the expression of LOX-5 and decreasing the LTB4 production in the tumor microenvironment.	97
			.
		HepG2 and MHCC97-L cells	Enhances Bax expression, formation of permeable transition pores, cytochrome C release to cytosol, and activation of the caspases 3 and 9 execution pathway.	98

BBR and Liver Cancer

Liver cancer, otherwise known as primary hepatic cancer or hepatocellular carcinoma (HCC), is considered one of the most prevalent and deadliest types of cancer universally, causing deaths with a significant percentage of humans annually.^6,99,100 In 2018, according to World Health Organization (WHO), liver cancer reportedly claimed about 782 000 human lives worldwide. It has greater predominance in males than in females, as well as high regional (Western and Middle Africa, Southeast and East Asia), and low regional (Eastern and Northern Europe, Western and South-Central Asia) incidence rates. ¹⁰¹ Nowadays, lung transplantation, surgery, and radiation therapies provide possible treatments for individuals with early detection.^102–108 Regardless of the therapeutic advancement, the treatment of liver cancer still remains burdensome due to the tendency of recurrence, even after curative treatment. Varied experimental studies have shown that BBR demonstrates enormous anti-cancer actions.^109,110

Effects of BBR on Overcoming Drug Resistance in Liver Cancer Treatment

BBR has demonstrated significant characteristics for overcome multidrug resistance, thus exhibiting its capacity in tumor chemotherapy. BBR regulates the neutrophil phenotype to maintain the sensitivity of cancer cells to doxorubicin, ¹¹¹ synergistically sensitizes human liver cancer cells to sorafenib, ⁸² and promotes the radio-sensitivity of hepatoma cells by inhibiting the Nrf2 pathway. ⁸³ Also, BBR and the Janus nanocarrier-based co-delivery of doxorubicin impairs chemotherapy-exacerbated HCC recurrence via inhibition of caspase-3-iPLA2-COX-2 signaling pathways. ¹¹² BBR combined with irradiation promotes anti-cancer actions through activation of the p38 MAPK pathway and ROS generation in human hepatoma cells.^84,113 Additionally, the combination of BBR and vincristine significantly impaired the growth and apoptotic induction in hepatoma cells. ¹¹⁴

Effects of BBR on Liver Cancer Metastasis Inhibition

BBR has been proven to possess positive attributes in the inhibition of tumor metastasis.^115,116 Matrix metalloproteinases (MMPs) break down the matrix tissue, thereby enabling the malignant cells to advance to the barrier of normal tissues and occupy the surrounding normal tissues and distant organs.

BBR suppresses Id-1 expression and inhibits the growth and development of lung metastases in HCC. ⁸⁵ BBR exerts a strong suppression on the invasion and migration of HCC cells through promoting PAI-1 and decreasing urokinase-type plasminogen activator (uPA). ⁸⁶

Mechanistic Effects of BBR on Liver Cancer

Oxidative Stress Regulation

Oxidative stress is a harmful process that can be an essential mediator of either impairment or destruction of cell structures and thus activates different disease conditions, namely cancer, diabetes, and neurological and cardiovascular diseases. Shukia et al demonstrated that BBR regulates oxidative stress through the promotion of reactive oxygen species (ROSs), and lipid peroxidation alongside the down-regulation of the actions of glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) expressions via the JUK signaling pathway. ⁸⁷ A traditional herbal medicine, Lagerstroemia speciosa (L.) Pers., containing of BBR, Gallic acid, and Corosolic acid, induces oxidative stress-mediated apoptosis associated with HepG2 cells through intrinsic and mitochondrial mechanisms. ¹¹⁷

Inflammation Reduction

Inflammation represents a common cause of different chronic diseases. BBR reduces the proliferation of liver cancer through an anti-inflammatory pathway. BBR reduces the protein expression levels of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2, and up-regulates the content ratio of arachidonic acid to prostaglandin E2 in the human hepatocarcinoma (HepG2) cells. ⁸⁸ BBR suppresses the phosphorylation of Akt, mTOR, and ERK, indicating inhibition of cell growth through the PI3K/Akt/mTOR and ERK/MAPK signal pathways. ⁸⁹ BBR induces G1-phase arrest accompanied by a decrease in cyclin D1, cyclin E, and cdc2 expressions. It inhibits cell growth through cell cycle arrest. In addition, BBR mediates proliferation and migration in HCC via the Wnt/β-catenin signaling pathway, ¹¹⁸ represses progression or growth of liver cancer cells via inhibiting glutamine uptake, ⁹⁰ and β-catechin translation involving 4E-binding protein. ⁹¹ BBR also exerts anti-proliferative effects against mitochondrial dysfunction-mediated apoptosis in HepG2 cells through down-modulation of the PI3K/Akt/mTOR mechanism, ⁸⁷ and impairs cell proliferation and migration in HCC via regulation of the Wnt signaling pathway. ⁸⁹ A traditional herbal remedy, known as DaHuangWan, constituted of BBR and costunolide, suppresses the proliferation of hepatoma cells through modulating the epithelial growth factor (EGF) mechanism. ¹¹⁹ Moreover, BBR can hamper cell proliferation of HepG2, Hep3B, and SNU-182 via promoting protein expression of tumor suppressor genes, like activating transcription factor 3 (ATF3). ⁹²

Apoptosis Activation

BBR potentiates the mitochondria-dependent pathway to activate apoptosis in human hepatocarcinoma (HepG2) cells. BBR restores the activation of caspase-3 and caspase-8 and releases mitochondrial membrane potential, cytochrome c, and cleavage of poly ADP-ribose polymerase (PARP), resulting in a decrease in the expression level of Bid and anti-apoptosis factor Bcl-XL. ⁹³ BBR remarkably activates the mRNA level or expression of FoxO1 and FoxO3 and inhibits their breakdown. Hence, BBR promotes the transcriptional effect of FoxO that is related to the anti-proliferation of tumors. Elevating FoxO transcriptional factors vigorously activates the level of the BH3-only protein Bim and induces the pro-apoptotic protein Bax and caspases, leading to mitochondria-mediated apoptosis. In addition, BBR activates apoptosis in liver cancer cells via inhibition of the AMPK-mediated mitochondrial/caspase pathway,^93–95 Akt-ASK1-P38MAPKs linked cascade, ⁹⁶ NF-kB p65 pathway, ⁹⁷ and the iPLA2/LOX-5/LTB4 signaling pathway. ¹²⁰ The combined use of BBR and evodiamine also improves the apoptosis of human HCC SMMC-7721 cells.^121,122 Furthermore, the use of Berberis lycium Royle in the apoptotic treatment of HepG2 cells, revealed a decrease in Bcl-2, independent of p53 Mrna, and an increase in CDK1 while suppressing CDK5, CDK9, and CDK10 mRNA expressions. ⁹⁸

Autophagy

Autophagy is described as a natural, conserved catabolic pathway, through which eukaryotic cells degrade or recycle internal components via a membrane trafficking mechanism. It also serves as a source of sustainable energy and biomolecules to the cells for proper maintenance of intracellular homeostasis during stressful conditions like a tumor microenvironment.

BBR activates autophagic cell death in HepG2 and MHCC97-L cells through suppression of the mTOR mechanism and Beclin-1 activation by increasing P38 MAPK and decreasing the activities of Akt signaling. ¹²³ BBR also induces the glucose-regulated protein 78 (GRP78) level via down-regulation of proteasomal and ubiquitination degradation, and activation of ATF6 cleavage, thereby resulting in cancer cell death and autophagy. ¹²⁴ BBR, obtained from Coptidis Rhizoma, triggered autophagic cell death via suppression of the PI3K/Akt/mTOR mechanism and elevation of ROS-mediated mitochondrial dysfunction in HCC Hep3B cells. ¹²⁵ In addition, BBR sensitized human HCC to ionizing radiation by obstructing cell cycle arrest and autophagy, resulting in senescence, ¹²⁶ triggered autophagic and apoptotic death in HepG2 cells through the activation of AMPK mechanism, ¹²⁷ and activated cell death in human hepatoma carcinoma cell line HepG2 through inhibiting the expression of CD147. ¹²⁸ Finally, BBR suppressed the viability, migration, and invasion capacity of HepG2 cells via the activation of pyroptosis (caspase-1 dependent programmed cell death) both in vitro and in vivo, which was impaired by caspase-1 inhibitor Ac-YVAD-CMK. ²⁸

In summary, BBR prevents cellular growth, proliferation, migration, autophagy, apoptosis, and cell cycle arrest processes in HCC cells via the inhibition of AMPK-mediated mitochondrial/caspase, arachidonic acid metabolic, NF-kB p65, iPLA2/LOX-5/LTB4, PI3K/Akt/mTOR, and Wnt signaling pathways. Figure 2 indicates the schematic illustration of BBR effects and mechanisms on liver cancer.

Figure 2.

Schematic illustration of berberine effects or mechanism in liver cancer.

The Enhancement of BBR Effects Through New Drug Formulation

Table 4 shows several formulation techniques particularly designed for tackling the limitations (poor intestinal absorption, poor pharmacokinetic, or poor bioavailability) associated with BBR administration, thereby promoting its anti-cancer efficacy.^29–45 These involve nanocarriers or nanoparticles of different surface charges and size with few targeting subcellular organelles like mitochondria. For instance, the experimental studies carried out by Khan et al, demonstrated a 14-fold elevation in the half-life of BBR in a mice model through poly (lactic-co-glycolic acid) (PLGA) nanoparticle BBR carriers, while the charged vitamin E-based amphiphilic mixed micellar vehicles provided a 30-fold enhancement in BBR pharmacokinetics. ²⁹ Also, the advancement in BBR cytotoxicity via preparation of liposomes has been discovered in vitro as well. ⁴⁶ This type of ongoing study show significant potential towards improving the efficacy of the most active BBR derivatives.

Table 4.

Formulation Techniques Designed to Promote the Bioavailability and Effectiveness of Berberine as an Anti-Cancer Agent.

No	Preparation	Attributes	Experimental model and effects	Ref
1	Nano-sized carbon nanocarrier-C60 fullerene (C60)	Water dispersions of noncovalent C60-Ber nano-complexes in 1:2, 1:1, and 2:1 molar ratios	Improves the intracellular uptake of BBR; higher anti-proliferative potential towards CCRF-CEM cells free—Berberine < 1:2 < 1:1 < 2:1 molar ratio preparations; induce caspase 3/7; cell cycle arrest at sub-G1 phase; activate apoptosis.	29
2	Cationic and anionic vitamin E-TPGS mixed polymeric phospholipid micellar vehicles	Lipid-based nanoparticles, amphiphilic mixed micelles composed of polymeric phospholipid conjugates and PEG-succinate ester of tocopherol.	Human prostate cancer cell lines (PC3 and LNPaC) promote apoptosis activation with a 30-fold potential improvement of pharmacokinetics.	30
3	Novel mitochondria targeting surface charge-reversal polymeric nanocarrier	Vitamin B6-oligomeric hyaluronic acid (OHA)-dithiodipropionic acid-BBR preparation; BBR conjugated with OHA and OHA further conjugated to B6. Micelles of 172.9 nm are formed by formulating conjugates with Cur-loaded nanoparticles.	Triggers cytotoxicity in vitro against PANC-1 cells and tumor proliferation in nude rats bearing PANC-1 cells xenograft; subcellular drug distribution indicates mitochondria as a target.	31
4	Planar side arm-tethered β-cyclodextrin encapsulation	Fluorenyl derivative of β-cyclodextrin used to encapsulate BBR.	Actively binds with duplex and G-quadruplex DNAs although its association with the cavity of β-cyclodextrin reduces the strength of binding.	32
5	Cationic γ-cyclodextrin derivative	A cationic derivative of γ-cyclodextrin synthesized via modification with propylenediamine; mucoadhesive with resistance to digestion by $\propto$ -amylase.	Localized in lysosomes with cytotoxicity twice higher than BBR in murine melanoma (B16-F10) and 4T1 cells.	33
6	PLGA nanocarrier	PLGA-doxorubicin conjugate utilized in BBR encapsulation.	Anti-proliferative against MDA-MB-231 and T47D breast cancer cell lines were observed with IC50 of 1.94  ±  0.22 and 1.02   ±   0.36 μM; changes mitochondrial permeability and arrest cell cycle at sub G1 phase; 14-fold up-regulates in the half-life of BBR in rats.	34
7	Self-carried berberine microrods	Particles prepared through mixing trimethylamine with BBR hydrochloride in DMSO to form about 20–100 μm in length and 5–20 μm width irregular size product.	Hepatocellular carcinoma (HepG2, SMMC-7721, Hep3B, H22 cells) and normal cell lines (HL-7702 cells, HUVEC cells, C2C12 cells, and H9C2 cells) utilized in cytotoxicity studies. With about 40 µg/mL IC50 value, about twice more selective than BBR in cancer cells.	35
8	Polyethyleneimine (PEI)-cholesterol (PC) berberine nanocarrier complexed with miR-122	BBR incorporated to PC with further electrostatic complex with miR-122; good drug loading (8.4%) and release (63.0) capacity of nanoparticles of about 146 nm.	Downregulates OSCC cells invasion and migration in transwell studies when compared with single-drug therapeutics.	36
9	BBR with PEGylated liposomal doxorubicin (PEG-lip-DOX)	BBR merged with polyethylene glycolated liposomal doxorubicin.	Suppresses the vascular endothelial growth factor (VEGF) expression in human umbilical vein endothelial cells (HUVECs); reduces (via i.v.) tumor proliferation in Meth A sarcoma-transplanted mice; effect stronger than BBR or PEG-lip-DOX alone.	37
10	Zinc oxide-based nanocarrier	BBR and zinc oxide (ZnO) combined through facile blending at the ratio of 39:61 to form 200–300 nm size nanoparticles.	Promotes anti-growth activity in A549 (human lung adenocarcinoma) cells; no obvious severe hepatotoxicity, hemotoxicity, and renal toxicity in rats by i.v.	38
11	Folic acid- and BBR-loaded silver nanomaterial (FA-PEG@BBR-AgNPs)	Encapsulating BBR on citrate-capped silver nanoparticles (AgNPs) through electrostatic interactions (BBR-AgNPs) followed through conjugation with polyethylene glycol-functionalized folic acid by hydrogen bonding interactions	Improves apoptosis in MDA-MB-231 breast cancer cells; activates ROS; regulates PI3K, AKT, Ras, Raf, ERK, VEGF, HIF1α, Bcl-2, Bax, cytochrome-c, caspase-9, and caspase-3; triggers tumor proliferation in vivo when given intravenously into MDA-MB-231 tumor-bearing athymic nude rat.	39
12	Hypoxia-specific chemo-targeting iron-oxide nanoparticle–berberine complexes	Hypoxic cell-sensitizer sanazole (SAN)-directed targeting of cytotoxic drug berberine and iron-oxide nanoparticle complexes.	Decrease tumor volume in rat bearing solid tumor in hind limb; elevates DNA damage; inhibits the levels of transcription of HIF-1α, VEGF, Akt, and Bcl2; promotes Bax and caspases expressions.	40
13	BBR-loaded Janus nanocarriers for magnetic field-improved therapy	Janus magnetic mesoporous silica nanoparticles (Fe3O4-mSiO2 nanoparticles): Fe3O4 head for magnetic targeting and a mesoporous SiO2 body for pH-dependent BBR delivery.	Magnetic field-activated endocytosis and pH-responsive drug release resulting in enhanced cytotoxicity against hepatocellular HepG2 carcinoma cells.	41
14	Dendrimer encapsulated and conjugated delivery of BBR	Dendrimer (G4 PAMAM) encapsulated and conjugated BBR formulations of 100–200 nm size; entrapment efficiency of 29.9% or percentage conjugation of 37.49%.	Higher drug payload in conjugation approach; sustained and efficient release pattern in vitro; higher anticancer action in vitro against MCF-7 and MDA-MB-468 breast cancer cells; no hemolytic effect ex vivo; enhanced pharmacokinetics in mice with about 2-fold advancement in half-life (t1/2).	42
15	Silver nanocarriers	Nanosize silver particles with BBR chloride.	Human tongue squamous carcinoma SCC-25–IC50 of 5.19 μg/mL; cell cycle arrest at G0/G1 phase; upregulation of Bax/Bcl-2 ratio gene expression.	43
16	Graphene oxide-based BBR nanocarrier	Electric-sensitive drug release and redox-sensitive graphene oxide nanocomposite loading BBR.	-	44
17	Solid lipid nanocarrier encapsulation.	Solid lipid nanoparticle (SLN) with a particle size of 81 nm and zeta potential of 28.67 ±  0.71 mV.	More cell growth inhibitory effect on MCF-7, HepG2, and A549 cancer cells than BBR; triggers cell cycle arrest, and apoptosis.	45
18	Liposomal BBR	Polyethylene glycol (PEG) with maximum encapsulation efficiency BBR as 14%.	2.5-times more active in diminishing the proliferation of HepG2 cells than BBR (IC50 of 1.67 μg/mL vs 4.23 μg/mL); activates apoptosis via the caspase/mitochondria-dependent signaling pathway; lower rate of elimination in both plasma and tissues; enhanced antitumor actions in vivo when tested in tumor xenograft rat bearing HepG2-triggered tumor.	46

Conclusion and Future Perspectives

In this review, we aimed to collect all information with regards to the various positive pharmacological and mechanistic effects of BBR for liver cancer treatment. The highlighted in vivo and in vitro experimental studies carried out for over a decade with the use of BBR for liver cancer treatment has proven the compound to be highly effective, safe, and a potent natural product, which could serve as a prospective choice as an herbal remedy in treating liver cancer. It also known to possess a wide spectrum of clinical usage against various diseases, such as polycystic ovary syndrome, NAFLD, CAD, and metabolic syndrome. However, a significant drawback is its hydrophilicity, known to be associated with the use of BBR in malignant or tumor treatment, thereby leading to poor bioavailability and low effective concentration. However, it has been proven that complexes of iron-oxide nanoparticles and the hypoxic cell sensitizer sanazole, in conjugation with BBR, represent a highly efficient improvement in the therapeutic specificity and bioavailability of BBR. However, more studies are required regarding the activities of BBR in preventing cellular growth, proliferation, migration, autophagy, apoptosis, and cell cycle arrest processes in HCC cells via different signaling pathways or mechanisms, so as to provide extensive analytical data to aid the conduction of clinical research in the future.