**2. Cell biology of cholesterol**

#### **2.1. De novo cholesterol biosynthesis**

Cholesterol is a 27-carbon and tetracyclic ring steroid that is catalyzed by a series of more than 26 separate enzymatic reactions in several subcellular compartments [20, 21]. The de novo biosynthesis can be considered as five major steps: (1) From acetyl-CoA to 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA): the acetyl-CoA can be derived from the oxidation of fatty acids or synthesized from cytosolic acetate precursors (metabolites or taken up from dietary or exogenous sources), and three acetyl-CoAs condense to form acetoacetyl-CoA by acetoacetyl-CoA acetyltransferases orthiolase and then HMG-CoA by HMG-CoA synthase.(2) The formation of mevalonate: HMG-CoA is reduced to mevalonate by HMG-CoA reductase, a rate-limiting andirreversible stepin themetabolicpathway thatproduces cholesterol andother isoprenoids. (3) From mevalonate to isopentenyl pyrophosphate (IPP): mevalonate is further converted to IPP through two phosphorylation steps and one decarboxylation step. This conversion is involved in seven different enzymes (mevalonate-3-kinase, mevalonate-5-kinase, mevalonate-3-phosphate-5-kinase, phosphomevalonate kinase, mevalonate-5-phosphate decarboxylase, mevalonate pyrophosphate decarboxylase, and isopentenyl phosphate kinase) via different avenues.(4) From IPP to squalene:three molecules ofIPP further condense to form a farnesyl pyrophosphate (FPP) and two molecules of FPP then condense to form squalene. The enzymes involved in the process are IPP isomerase, farnesyl-diphosphate synthase, and squalene synthase. (5) From squalene to lanosterol to cholesterol: the oxidation of squalene by squalene epoxidase forms 2,3-oxidosqualenewhichis further cyclizedto lanosterol bysqualene oxidocyclase. Lanosterol is finally converted to cholesterol by a series of demethylations, desaturations,isomerizations, andreductions.Demethylation reactionsproduce zymosterol as an intermediate and further converted to cholesterol by at least two pathways that differ in the order of the desaturations, isomerizations, and reductions (**Figure 1**) [22–27].

**Figure 1.** Scheme of the cholesterol biosynthesis pathway. (1) Thiolases or acetyl-coenzyme A acetyltransferases, (2) hydroxy-3-methylglutaryl-CoA synthase, (3) hydroxy-3-methylglutaryl-CoA reductase, (4) mevalonate-3-kinase or mevalonate-5-kinase, (5) mevalonate-3-phosphate-5-kinase or phosphomevalonate kinase, (6) mevalonate-5-phosphate decarboxylase, (7) mevalonate pyrophosphate decarboxylase, (8) isopentenyl phosphate kinase, (9) isopentenyl pyrophosphate isomerase, (10) farnesyl-diphosphate synthase, (11) squalene synthase, (12) squalene monooxygenase or squalene epoxidase, and 19 reactions are included multiple demethylations, desaturations, isomerizations, and reductions.

#### **2.2. Cholesterol homeostasis**

Due to the key physiologicalroles that cholesterol plays,the circulating and cellular cholesterol levels in our body are tightly regulated by a physiological balance of cholesterol biosynthesis, cholesterol catabolism, cholesterol transportation (influx and efflux), dietary cholesterol absorption, andcholesteroldepletion.Higher cholesterol, alsoknownashypercholesterolemia, is a risk factor for a variety of human diseases such as cardiovascular diseases, dyslipidemia, Alzheimer'sdisease,HIVdyslipidemia, chronic inflammation, anddevelopingdiabetes.Earlier data also indicates that accelerated cholesterol metabolism and elevated cholesterol levels contribute tothehallmarksof cancerdevelopmentandmalignanttransformation[9–15].Cancer cells need excess cholesterol and intermediates of the cholesterol biosynthetic pathway to maintain a high level of cell growth and proliferation. Meanwhile, cholesterol is capable of regulating multiple signaling pathways involved in carcinogenesis, cancer cell migration, and tumor progression and is also involved in chemosensitivity and chemotherapy resistance of cancer cells [9–19]. It is very important to understand cholesterol as an important factor contributing to carcinogenesis and tumor progression and to elucidate the regulation of cholesterol metabolism as a new strategy for searching cancer prevention and therapy drugs.

Cholesterol is a 27-carbon and tetracyclic ring steroid that is catalyzed by a series of more than 26 separate enzymatic reactions in several subcellular compartments [20, 21]. The de novo biosynthesis can be considered as five major steps: (1) From acetyl-CoA to 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA): the acetyl-CoA can be derived from the oxidation of fatty acids or synthesized from cytosolic acetate precursors (metabolites or taken up from dietary or exogenous sources), and three acetyl-CoAs condense to form acetoacetyl-CoA by acetoacetyl-CoA acetyltransferases orthiolase and then HMG-CoA by HMG-CoA synthase.(2) The formation of mevalonate: HMG-CoA is reduced to mevalonate by HMG-CoA reductase, a rate-limiting andirreversible stepin themetabolicpathway thatproduces cholesterol andother isoprenoids. (3) From mevalonate to isopentenyl pyrophosphate (IPP): mevalonate is further converted to IPP through two phosphorylation steps and one decarboxylation step. This conversion is involved in seven different enzymes (mevalonate-3-kinase, mevalonate-5-kinase, mevalonate-3-phosphate-5-kinase, phosphomevalonate kinase, mevalonate-5-phosphate decarboxylase, mevalonate pyrophosphate decarboxylase, and isopentenyl phosphate kinase) via different avenues.(4) From IPP to squalene:three molecules ofIPP further condense to form a farnesyl pyrophosphate (FPP) and two molecules of FPP then condense to form squalene. The enzymes involved in the process are IPP isomerase, farnesyl-diphosphate synthase, and squalene synthase. (5) From squalene to lanosterol to cholesterol: the oxidation of squalene by squalene epoxidase forms 2,3-oxidosqualenewhichis further cyclizedto lanosterol by squalene oxidocyclase. Lanosterol is finally converted to cholesterol by a series of demethylations, desaturations,isomerizations, andreductions.Demethylation reactionsproduce zymosterol as an intermediate and further converted to cholesterol by at least two pathways that differ in the

order of the desaturations, isomerizations, and reductions (**Figure 1**) [22–27].

**2. Cell biology of cholesterol**

108 Cholesterol Lowering Therapies and Drugs

**2.1. De novo cholesterol biosynthesis**

Cholesterol is a vital lipid and plays well-described biochemical roles and diverse functions at cellular level [1–3]. The homeostasis of cholesterol is among the most intensely regulated processes in our body. High cholesterol is a risk factor to numerous pathologies such as cardiovascular disease, atherosclerosis, dyslipidemia, and neurodegenerative diseases and is associated with the development of diabetes and cancer. Cholesterol homeostasis is achieved through intricate mechanisms involving biosynthesis, catabolism, dietary absorption, transportation(influx or efflux), anddepletion(**Figure 2**)[28–32]. Slightly less thanhalf of cholesterol in our body derives from de novo biosynthesis every day. The liver is the dominant site of cholesterol biosynthesis, and in vivo liver cholesterol production has been estimated at 1–2 g/ day. Cholesterol is synthesized in liver and then secreted as circulating lipoproteins into bloodstream. The intestine and skin are also very important for cholesterol synthesis [33–35]. Although the majority of cholesterol sources comes from cholesterol biosynthesis, it is under feedback regulation. The absorption of cholesterol mainly derives from three sources: diet, bile, and intestinal epithelial sloughing. The average intake of cholesterol in the Western diet is approximately 300–500 mg per day. Bile is estimated to contribute nearly 800–1200 mg of cholesterol per day to the intraluminal pool. A third source of intraluminal cholesterol comes from the turnover of intestinal mucosal epithelium, which provides roughly 300 mg of cholesterol per day [36]. In cholesterol catabolism, the conversion of cholesterol into excretable bile acids represents themostrelevantmechanismofirreversible eliminationof cholesterolfromthe body, which plays a key role in hepatic and systemic cholesterol homeostasis. Under physiological conditions, approximately 300–400 mg of cholesterol is disposed in the liver daily [37]. Because peripheral cells do not catabolize the cholesterol molecule, there are two distinct mechanisms for maintaining cellular cholesterol homeostasis. One is the nonspecific classical pathway mediated by physicochemical diffusion of cholesterolthrough the aqueous phase and the other is cholesterol esterification on high-density lipoprotein (HDL) by lecithin: cholesterol acyltransferase reaction [38, 39]. The reaction is initiated by the interaction of lipid-free or lipidpoor apolipoproteins with cellular surface resulting in the assembly of HDL particles with phospholipid and cholesterol as well as extracellular cholesterol esterification mainly on HDL [40]. Furthermore, changing dietary style to control cholesterol absorption and using pharmaceutical drugs to inhibit several key enzymes in cholesterol synthesis can also significantly reduce the level of cellular cholesterol. All ofthese pharmaceutical drugs and dietary style have been commonly used for keeping a healthy life and preventing heart disease [41–44].

**Figure 2.** Cholesterol homeostasis and functions. Cholesterol homeostasis is tightly regulated in our body and can be achieved through intricate mechanisms involved in biosynthesis, dietary absorption, transportation (influx or efflux), catabolism, and depletion. The functions of cholesterol are composed of distinct membrane, control membrane fluidity and protein recruitment, produce steroid and oxysterol, and are involved in cell signaling to regulate cell growth, proliferation, and migration.

#### **2.3. Biological functions of cholesterol**

cholesterol biosynthesis, and in vivo liver cholesterol production has been estimated at 1–2 g/ day. Cholesterol is synthesized in liver and then secreted as circulating lipoproteins into bloodstream. The intestine and skin are also very important for cholesterol synthesis [33–35]. Although the majority of cholesterol sources comes from cholesterol biosynthesis, it is under feedback regulation. The absorption of cholesterol mainly derives from three sources: diet, bile, and intestinal epithelial sloughing. The average intake of cholesterol in the Western diet is approximately 300–500 mg per day. Bile is estimated to contribute nearly 800–1200 mg of cholesterol per day to the intraluminal pool. A third source of intraluminal cholesterol comes from the turnover of intestinal mucosal epithelium, which provides roughly 300 mg of cholesterol per day [36]. In cholesterol catabolism, the conversion of cholesterol into excretable bile acids represents themostrelevantmechanismofirreversible eliminationof cholesterolfromthe body, which plays a key role in hepatic and systemic cholesterol homeostasis. Under physiological conditions, approximately 300–400 mg of cholesterol is disposed in the liver daily [37]. Because peripheral cells do not catabolize the cholesterol molecule, there are two distinct mechanisms for maintaining cellular cholesterol homeostasis. One is the nonspecific classical pathway mediated by physicochemical diffusion of cholesterolthrough the aqueous phase and the other is cholesterol esterification on high-density lipoprotein (HDL) by lecithin: cholesterol acyltransferase reaction [38, 39]. The reaction is initiated by the interaction of lipid-free or lipidpoor apolipoproteins with cellular surface resulting in the assembly of HDL particles with phospholipid and cholesterol as well as extracellular cholesterol esterification mainly on HDL [40]. Furthermore, changing dietary style to control cholesterol absorption and using pharmaceutical drugs to inhibit several key enzymes in cholesterol synthesis can also significantly reduce the level of cellular cholesterol. All ofthese pharmaceutical drugs and dietary style have

been commonly used for keeping a healthy life and preventing heart disease [41–44].

**Figure 2.** Cholesterol homeostasis and functions. Cholesterol homeostasis is tightly regulated in our body and can be achieved through intricate mechanisms involved in biosynthesis, dietary absorption, transportation (influx or efflux), catabolism, and depletion. The functions of cholesterol are composed of distinct membrane, control membrane fluidity and protein recruitment, produce steroid and oxysterol, and are involved in cell signaling to regulate cell growth, pro-

liferation, and migration.

110 Cholesterol Lowering Therapies and Drugs

Disruption to cholesterol homeostasis leads to a variety of diseases such as coronary heart disease, atherosclerosis, and metabolic syndrome as well as cancer[9–19, 45–51]. This indicates that cholesterol plays a crucial role in the regulation of cellular function (**Figure 2**). In the cells, cholesterol is mandatory for cellular growth and serves as one of the necessary building blocks for new membranes demanded by dividing cells during proliferation. Cell membranes have been recognized as heterogeneous structures composed of distinct membrane microdomains with different proteins and lipids. Lipid rafts, cholesterol-rich domains, play an important platform as a signaling station for many cellular processes, including membrane sorting and trafficking, cell polarization, and signal transduction [52–56]. Cholesterol promotes cell proliferation by inducing the activation of the AKT and/or the ERK signaling pathway as well as Ca2+ channel [57–60] and cell migration by increasing the activity of calpain that is also Ca2 <sup>+</sup> dependent [61, 62] and is also involved in Hedgehog processing, diffusion, and reception [63, 64]. Cholesterol can be converted to steroid hormones which activate nuclear receptors and thus help to control metabolism, inflammation, immune functions, salt and water balance, the development of sexual characteristics, and the ability to withstand illness and injury [65, 66]. Meanwhile, the metabolites of cholesterol such as hydroxycholesterols play multiple biological functions in the body [67, 68]. Cholesterol also contributes to chemotherapy resistance which leads to treat failure [11–14]. Taken together, cholesterol is tightly associated with cancer cell growth, proliferation and therapy.
