Effect of Modified HIF-1 α Linked to Carbonic Anhydrase IX Inhibitor and Glycosylated Cisplatin on Solid Tumors: Short Review

Many cancer cells in solid tumors are hypoxic or pseudohypoxic and create acidic environment for malignancy progression. Under low oxygen conditions (hypoxia), hypoxia-inducible factors (HIFs) play pathological roles in cancer cell survival and spreading. HIF regulates several genes such as genes of glucose transporters that enhance anaerobic glycolysis, angiogenesis, eryt-hropoiesis and carbonic anhydrase IX (CA-IX). CA-IX is a cell-surface glycoprotein that catalyzes the hydration of CO 2 to protons and bicarbonate ions (respiratory acidification). This process is involved in adaptation to acidosis and implicated in cancer progression. Therefore, CA-IX inhibitors (such as sulfonamide-based compounds) showed hoping results in reduction malignancy progression. The article aims to reversal the malignant hypoxic environment in solid tumors to create a condition of weakness within the cancer for further focused cisplatin potency. This article suggests the use of modified synthesized HIF as a drug delivery molecule for both carbonic anhydrase IX inhibitor and glycosylated cisplatin that damages the DNA of malignant cell. HIF molecule has high affinity to bind with CA IX-expressing malignant cells, which is followed by cell entrance via endocytosis. Once the HIF-Cisplatin-CA-inhibitor complex enters the cell, the carbonic anhydrase inhibitor will improve the cellular pH that makes the environment unsuitable for HIF 1α function and it may be ubiquitinated. So, the raise in target genes transcription will be arrested. On the other hand, once the synthetized HIF is degraded, the cisplatin molecules will be released inside the malignant cell and start to damage its DNA. This approach may be a good solution for many solid tumors.


HIF-1α Activity Differences in Normoxic and Hypoxic Conditions
The characteristic features of many tumor cells are that they are hypoxic and acidic compared with its normal cells of the same type. When the oxygen is decreased, hypoxia-inducible factors (HIFs) play physiological and pathological roles in response to decreased oxygen levels (hypoxia). HIFs are dimeric molecules that act as transcriptional factors and degraded in the presence of oxygen.
HIF-1α synthesis occurs in the cytosol and it is degraded by the 26 s proteasome ubiquitination system in normoxic environment [1].
During hypoxia, HIF-1α structure is kept intact and binds with HIF-1β (also known as ARNT, aryl-hydrocarbon-receptor nuclear translocator) [2] besides specific coactivators such as CBP/p300 that bind with HIF-1α for activation of HIF translocation and transcription [3]. Then HIFα is translocated to the nucleus, especially to conserved sequences (RCGTG) in the promoter regions of HIF-regulated genes (Hypoxia response elements (HRE) [4]. Hypoxia response elements are the transcriptional targets of HIFα, that are essential for metabolic cellular consequences in the hypoxic condition [5], which preserve the cellular viability such as carbonic anhydrase-IX (CA-IX) that controls intracellular and extracellular pH for the purpose of malignancy spread, dissemination and growth [2], glucose transporter-1 (GLUT-1), which is a transmembranous glucose transporter [3]. GLUT-3, hexokinase 2, lactate dehydrogenase [6] and vascular endothelial growth factor (VEGF) that has important role in endothelial cell proliferation to form new capillaries and tumor blood vessels in response to hypoxia.
The final aim of HIFα is to give the malignant cells the ability for anaerobic glycolysis and angiogenesis, also it helps the malignant cells to be adapted and alive under stressful hypoxic conditions [5] (Figure 1).

Regulation of HIF Function
The HIF function is regulated by some biological reactions such as: 1) S-nitrosylation of cysteine 800: This is the key factor for gene transcription con-  proteome for proteasomal degradation [10]. The second is asparagine-targeting factor inhibiting HIF (FIH) enzyme which is O 2 dependent asparaginyl hydroxylase [11]. FIH enzyme inhibits HIFα binding to CREB-binding protein (CBP/p300) which suppresses the trans-activated target genes without affection HIFα structural stability [12]. PHDs and FIH hydroxylation function is suppressed in case of hypoxia, in result HIFα will be accumulated after its stabilization and protection against ubiquitination by pVHL [8] [9] ( Figure 1).

Role of Carbonic Anhydrase in Cancer Cell pH Control and Its Relationship with Hypoxia
CA-IX is a glycoprotein (transmembranous) that motivates the reversible hydration of CO 2 to H 2 CO 3 during glycolysis. Also it is present in high concentrations in many cancer cell types and has predictive value of hypoxia in many neoplasms, which have bad prognosis [13]. Intracellular carbonic anhydrase inhibitors isoforms were found to be present in cancer types in high concentrations like colorectal HCT116, Bladder RT112 and colorectal HT29, moderate concentrations in fibrosarcoma HT1080. Malignant neoplasms of low levels of carbonic anhydrase inhibitors are aggressive forms [14].
CA-IX is important for cancer cell signaling, tumor formation, acidification and metastasis, so their inhibition was a target of studies in malignant neoplasms treatment. CA-IX inhibitor showed hoping results in reduction malignancy progression in vitro and in vivo [15]. Sulfonamide based compounds are the most Journal of Biosciences and Medicines successful CA IX inhibitors.
During hypoxia, malignant cells are directed towards metabolic shift of rapid aerobic glycolysis to yield lactic acid even in the presence of oxygen, which is known as the "Warburg Effect" [16]. Export lactic acid and free protons from malignant cells will lower pHe (pH extracellular) to (6.5 -7.1) with mild decrease in pHi (pH intracellular) to be 7.2 or more [17]. Fast shift of protons transport in and out the cell membrane will equilibrate the metabolic state of the malignant cell in case of any changes [18].
Low pHe will create suitable environment for metastasis and cancer progression [19] by enhancing extracellular matrix (ECM) reorganizational proteasese like matrix metalloproteinases (MMP) and cathepsin [20]. pHi 7.2 or more will predisposed to de novo actin filament formation by activation actin binding proteins such as cofillin, villin, profilin, twinfilin, and talin that preserve tumor cell survival [21] [22] [23] Also conversion of glucose to lactic acid will produce CO 2 , which is hydrated to carbonic acid H 2 CO 3 , then dissociated to 3 HCO − and H + (the source of respiratory acidification) [24]. That previous reaction is the function of carbonic anhydrase IX. 3 HCO − (bicarbonate) and H + (protons) are carried by bicarbonate transporter proteins to blood or tumor cell, in that way the control of pH in cancer cells is done. Also intracellular tail of CA-IX activates many cancer signaling cascades including HIFα. This is performed through the reaction of its phosphorylated tyrosine residue with the regulatory subunit of phosphoinositol 3 kinase (PI3K) that has a role in HIF activation [25].
Also it stimulates angiogenesis, invasion, and metastasis. It is associated with aneuploidy, increased mutation rate, survival and cell migration [27] [28]. Carbonic anhydrase IX has special mechanisms for malignancy progression like acting as an adhesion molecule during cell attachment in solid tumors [29]. It also stimulates angiogenesis by endothelial cells migration [30]. CA-IX induced acidosis releases cells in the form of clusters from the primary tumor into the circulation freely without adhesions in between to augment metastasis [31].
2) SUMO-1 (small ubiquitin-related modifier) modification at Lys (391, 477, 532, 538 and 547) residues: This stabilizes HIF-1 alpha and enhances its transcriptional activity. (VHL)-E3 ubiquitin ligase binds the target protein and the E2 catalyzes ubiquitin transfer to lysine residues K532, K538 and K547. Only one of these lysine residues need to be intact for full ubiquitination to occur. So, the desumoylated HIF molecule is susceptible to degradation [33].

Use of HIF as a Drug Delivery Molecule
In the malignant cell there is a cycle started from pVHL silencing, inhibition of the hydroxylase activity (PHDs) and FIH, lead to accumulation of HIF-1α, then induction of hypoxic environment. These conditions activate the carbonic anhydrase IX and other enzymes that induce Warburg effect, which results in more acids production from excessive glycolysis. In a result, there will be lowering pH intracellular and extracellular (pHe < pHi). This leads to more accumulation and production HIF-1α that increases malignancy progression and the cycle is repeated.
So, breaking of this cycle is our aim. This can be achieved through two stages: 1) reversal of the acidic environment by carbonic anhydrase IX inhibitor, and 2) damaging malignant cell (that secrete HIF-1α and CA-IX) DNA genome by platinum containing cytotoxic drug e.g. cisplatin in its glycosylated form. Carbonic anhydrase inhibitor and glycosylated cisplatin will be linked on modified synthetized HIF-1α. Synthetized HIF-1α from microbial culture media will be modified by adding certain enzymes that promote and inhibit post-translational modifications within the protein. Also those modifications will enable us to attach some therapeutic agents by the way that does not change its 3rd structure.
So, the modified HIF-1α molecule will be used as a drug delivery molecule in ineffective and easily ubiquitinated structure.
Use of modified HIF-1α as a drug carrier molecule may be shocking, but the affinity of modified HIF-1α to hypoxic malignant cells expressing CA IX is fairly good. By that way, the uptake of HIF-1 alpha will be easier after CA inhibitor linkage to CA IX expressing malignant cells that secrete HIF-1 alpha. Pep-Journal of Biosciences and Medicines tide-CA inhibitor can enter malignant hypoxic cells by the way of endocytosis [35].
On just entry the cell, carbonic anhydrase inhibitors will improve the cellular pH that makes the environment unsuitable for HIF-1α (that is formed by the malignant cells) functioning or it may be ubiquitinated, in a result, it will be accumulated within the cytoplasm not translocated to the nucleus. So, the raise in target genes transcription will be arrested. On the other hand, once the synthetized HIF-1 alpha is degraded the glycosylated cisplatin will be released intracellular. Glycosylated cisplatin is converted to its active form by reducing enzymes e.g. glutathione and ascorbic acid [36], then forming platinum DNA-adducts.
The end result is damaging malignant cell DNA, which is the factory of HIF-1α and CA-IX enzyme specifically.

1) Microbial production of modified HIF-1 alpha:
HIF-1α is produced under aerobic (normoxic) conditions (normoxic conditions are favorable for proline 564 hydroxylation and preserving serine residues without modifications) from E. coli strain K 12, which contains flavohemoglobin [37]. Flavohemoglobin is involved in NO detoxification in aerobic process for denitrosylation [38]. Denitrosylation of cysteine 800 is important to be attachment site to CA-IX inhibitor such as functionalized heterocyclic primary sulfonamide with NO moiety. SIR 2 protein is an enzyme which will be injected in the form of liposomal packaging into the culture cells. Its role is to deacetylate lysine 709 mainly that will make it of increased binding affinity for prolyl hydroxylase 2 and increased HIF-1α hydroxylation and ubiquitination later on when it is used as a therapeutic molecule by VHLp [34]. Also SENP2 protease (desumoylating enzyme) that catalyze two essential functions in the SUMO pathway will be used also in the form of liposomal packaging (liposomes are used as an efficient method to entrap enzymes and transport them into the cells. In that stage, we preferred in vitro O-glycosylation to avoid change HIF-1α 3D-structure; especially O-glycosylation introduces novel bonding interactions to the protein and preserves its integrity [42]. Also glycosylation preserves protein folding by destabilization of its unfolded condition in noticeable manner than stabilization the folded structure [43]. Choosing glycosylated cisplatin is done because it is a prodrug in inactive form. It will be more efficient than cisplatin as it will resist malignant defense mechanism and will reduce the possible side effects. Other advantages of glycosylated cisplatin are improved bioavailability. So it is easier to enter malignant cells before reduction to platinum (II) forming DNA-platinum adducts damaging malignant DNA nucleotide [36].
This in-vitro S-nitrosylation of HIF-1 alpha doesn't change its 3D-structure and it is a very important step because HIF-1 alpha cysteine 800, if not blocked by carbonic anhydrase inhibitor or any other compounds, it can be S-nitrosylated inside the malignant cell by histone deacetylases or by other different mechanisms e.g.
GADPH-mediated trans-nitrosylation enzymes, hence activated to its carcinogenic form. Linking CA-IX inhibitor will direct the synthetized HIF-1α with glycosylated cisplatin to enter malignant cells that express CA-IX, which is commoner and regarded as a marker for HIF-1α (CA-IX synthesis is a result from HIF-1α transcriptional activity [2]. Sulfonamide Carbonic Anhydrase IX inhibitor, with NO moiety: 2-methoxy-4-(3-(4-(nitroxy)butoxy)-3-oxoprop-1-en-1-yl)benzyl4-sulfamoylbenzoate. Journal of Biosciences and Medicines In this hypothesis, sulfonamide CA IX inhibitor and cisplatin is linked to HIF-1α ( Figure 3) (that has familial structure to hypoxic malignant cells) ( Figure   4(a)), so that this inhibitor is directed specifically towards malignant hypoxic cell membranes exerting their pH modulating effect and acidity reversal action, then selectively induces CA-IX-related endocytosis of HIF-1α alpha-glycosylated cisplatin intracellular. Then synthetized HIF-1α will face unsuitable intracellular pH, so it will be ubiquitinated (Figure 4(b)) especially after the previously modified post-translational modifications have been done (Figure 3). The final result is release of 6 glycosylated cisplatin platinum-IV molecules (prodrug) after HIF 1 alpha ubiquitination intracellular that will be reduced to platinum II (the active form) by the effect of ascorbic acid and gluthathione that are intensively present in malignant cells [36] making DNA adducts and destroying malignant cell genome (Figure 4(c)).

Conclusion
Expected scenario when HIF-1α-CA-IX Inhibitor-glycosylated cisplatin complex used as a cytotoxic drug in solid tumors e.g. hepatoma and renal cell carcinoma (hypoxic or pseudo-hypoxic tumor), it will be transferred easily intracellular (inside malignant cells only) because HIF-1α acts as a targeting component towards malignant cells that express CA-IX. HIF-1α will enter the hypoxic malignant cells by endocytosis. Carbonic anhydrase IX inhibitor will modify intracellular pH towards the normal range (less acidic and less hypoxic), which will alter the malignant cell environment to be unsuitable for HIF-1α functioning especially after blocking its Cys 800, which is nitrosylated and blocked by binding to carbonic anhydrase inhibitor. This is in addition to blocking of the serine residues and preventing its phosphorylation when glycosylated with glycosylated cisplatin. By that way HIF-1α will be inactive and easily degraded, especially after desumoylation and deacetylation of lysine residues, which are the main route for  its ubiquitination. This can be performed directly through the available lysine residues 532, 538 and 547 by the ubiquitin attachment or indirectly by the hydroxylated proline 564. After HIF-1α intracellular ubiquitination, the six glycosylated cisplatin molecules will be released and reduced by glutathione and ascorbic acid to its active form, which in turn will form DNA-platinum adducts mispairing malignant cells nucleotide. Cisplatin will have augmentation effect on CA-IX inhibitor in comparison to usage CA-IX inhibitor only because it will destabilize the overproduced genes that are responsible for synthesis glycolysis enzymes, CA IX and angiogenesis proteins. Those genes are responsible for creation the acidic pH that resists CA-IX inhibitor. That new molecule can be used prior to TACE (Transarterial Chemotherapy) and surgery in large solid tumors like hepatoma and renal cell carcinoma.