Pharmaceutical Biotechnology - Lecture 3 Summary

Multistep Carcinogenesis & Therapy Targets

Based on the carcinogenic process and the hallmarks of cancer, therapy targets can be identified. Carcinogenesis involves defects in terminal differentiation, growth control, programmed cell death resistance, and sustaining proliferative signaling. It also includes unlocking phenotypic plasticity, deregulation of cellular metabolism, genomic instability, and mutation, activation of proto-oncogenes, inactivation of tumor suppressor genes and genomic stability genes, epigenetic changes, evading growth suppressors and immune destruction, enabling replicative immortality, tumor-promoting inflammation, inducing vasculature, polymorphic microbiomes, and activating invasion and metastasis.

Interference with Carcinogenesis Using Small Molecules

Small molecules can interfere with carcinogenesis in several ways including the inhibition of DNA replication.

Inhibition of Cancer Cell Proliferation

Cancer is characterized by mitotic cell proliferation. Mitosis is a somatic cell division that continues indefinitely in cancer cells. Mitosis involves several stages, including prophase (early and late), metaphase, anaphase (early and late), and telophase, resulting in two cells with the same structures and chromosome number as the parent cell.

Cancer Chemotherapy

Chemotherapy is the use of drugs to treat diseases, particularly cancer. The mechanism of action and drug production vary for each drug.

Biotechnology in Chemotherapy Production

Biotechnological methods are used to create chemotherapeutic drugs when extraction from the original source or in vitro synthesis is time-consuming and/or expensive. Biotechnology has improved drug production technology, drug testing, identification of novel drug targets, and new approaches to therapy, such as precision oncology.

Cancer Therapy - Activation of Immune System

Cancer therapy involves activating the immune system, where macrophages, T-cells, and monocytes release pro-inflammatory cytokines and activate apoptotic pathways, leading to cell death. Chemotherapeutic drugs can also target mitochondria and microtubules.

Effects of Chemotherapeutic Drugs in Cancer Cells

Mitochondria

Drugs like oxaliplatin, cisplatin, vincristine, and paclitaxel can:

• Activate apoptosis.
• Alter the function of the respiratory chain, leading to altered mitochondrial function.
• Inhibit mitochondrial DNA (mDNA) replication/transcription, causing increased reactive oxygen species (ROS).

Microtubules

• Vincristine inhibits microtubule aggregation.
• Paclitaxel inhibits microtubule disaggregation.
• These actions alter the function of microtubules, leading to cell arrest.

Nucleus

• Oxaliplatin and cisplatin inhibit DNA replication and mRNA transcription.
• This leads to cell arrest and cell death.

Paclitaxel (PTX) and Vincristine

Paclitaxel (PTX) is a potent antitumor alkaloid used to treat various cancer types. It was approved by the FDA in 1992 as a broad-spectrum drug. Paclitaxel naturally exists in low amounts in the inner bark of Taxus species. Its global demand increases by about 6–10% annually. The mechanism of action of vincristine sulfate inhibits microtubule formation in the mitotic spindle, arresting dividing cells at the metaphase stage. Central nervous system leukemia has been reported in patients undergoing therapy with vincristine sulfate. Vincristine is naturally extracted from Catharanthus roseus. Alternate production methods include semi-synthesis coupling and stereocontrolled total synthesis. Liposome encapsulation enhances efficacy and reduces neurotoxicity.

Paclitaxel and Vinca Alkaloids

Paclitaxel (PTX) binds to $β$-tubulin and inhibits the breakdown of microtubules, leading to their accumulation and non-functionality, blocking cell division in the G2/M phase and causing apoptotic cell death. Vincristine, a vinca alkaloid, also binds to $β$-tubulin and inhibits the aggregation of microtubules, leading to G1 phase arrest and apoptotic cell death.

Cancer Drugs Acting in the Nucleus and Mitochondria

Certain cancer drugs exhibit their effects by targeting the nucleus and the mitochondria of cancer cells.

Cisplatin, Carboplatin, and Oxaliplatin & Fluorouracil (5-FU)

Platinum Compounds

Cisplatin, carboplatin, and oxaliplatin are platinum compounds administered intravenously in chemotherapy treatment. Platinum analogs are antineoplastic alkylating agents used to treat approximately half of the patients receiving chemotherapy. They cause crosslinking of DNA as monoadduct, interstrand crosslinks, intrastrand crosslinks, or DNA protein crosslinks, primarily acting on the adjacent N-7 position of guanine, forming a 1,2 intrastrand crosslink. This crosslinking inhibits DNA repair and/or DNA synthesis.

Fluorouracil(5-FU)

5-FU was developed in 1957. It is catabolized to inactive metabolites by dihydropyrimidine dehydrogenase (DPD). Only a small percentage mediates cytotoxic effects on tumor cells and normal tissues by inhibiting DNA synthesis, RNA processing, and function. The 5-FU metabolite, fluorodeoxyuridine monophosphate (FdUMP), forms a ternary complex with thymidylate synthase (TS) and 5,10-methylene tetrahydrofolate ($CH_2THF$), inhibiting DNA synthesis.

Cytotoxicity and Side Effects

Cytotoxicity is higher for cisplatin and oxaliplatin compared to carboplatin, with apoptosis as the preferred mode of cell death. Platins can cause over 40 specific side effects, including neurotoxicity manifested by peripheral neuropathies like polyneuropathy.

Mechanism of Action of Fluorouracil and Alkylating Platinum Agents

Fluorouracil

Fluorouracil inhibits DNA synthesis and RNA processing. The 5-FU metabolite, fluorodeoxyuridine monophosphate (FdUMP), forms a ternary complex with thymidylate synthase (TS) and 5,10-methylene tetrahydrofolate ($CH_2THF$), inhibiting DNA synthesis.

Alkylating Platinum Agents

Platinum-based antineoplastic agents cause crosslinking of DNA as monoadduct, interstrand crosslinks, intrastrand crosslinks, or DNA protein crosslinks. They primarily act on the adjacent N-7 position of guanine, forming a 1,2 intrastrand crosslink. The resultant crosslinking inhibits DNA repair and/or DNA synthesis.

Small Molecule Entry into Cells

A small molecular drug needs to enter a cell to exert its action.

Types of Membrane Transport

Membrane transport includes diffusion, facilitated diffusion, active transport, and passive transport.

Platinum Drug Accumulation Mechanisms

Platinum (Pt) drug accumulation involves passive diffusion and channel-mediated mechanisms. Channel-mediated mechanisms include:

• VRAC (volume-regulated anion channel).
• LRRC8 (leucine-rich repeat-containing 8).
• CTR1 (copper transporter 1).
• OCT1/2/3 (organic cation transporter 1/2/3).
• OCTN1/2 (organic cation/carnitine transporter 1/2).
• GSTP1 (glutathione S-transferase pi 1).
• MRP2 (multidrug resistance-associated protein 2).
  Unconjugated oxaliplatin enters through solvent-accessible cysteine.

Details of Platinum Drug Accumulation Mechanisms

The hexameric volume-regulated anion channel (VRAC) is composed of leucine-rich repeat-containing 8 (LRRC8) subunit proteins and is involved in osmolytes exchange, regulating cell volume. In cancer cells, VRAC mediates the uptake of cisplatin and carboplatin, and the loss of subunits LRRC8A or LRRC8D increases tumor cell resistance. Fifty percent of cellular platinum (Pt) uptake is attributed to VRAC, with the remainder occurring through passive diffusion or other channel-mediated uptake mechanisms.
Other transporters, such as copper transporter 1 (CTR1), organic cation transporter 1 (OCT1), OCT2, OCT3, organic cation/carnitine transporter 1 (OCTN1), and OCTN2, import Pt-based agents into the cell, and their loss increases resistance. Glutathione S-transferase pi 1 (GSTP1) mediates binding of Pt-based agents to free cysteines and subsequent export via multidrug resistance-associated protein 2 (MRP2), which is another resistance mechanism. MRP2 also exports unconjugated oxaliplatin from the cell.

Pt Drug Compounds and Formulations

There is ongoing research to find platinum (Pt)-based agents or formulations to reduce patient toxicities and tackle resistance development. Current Pt drugs’ DNA binding modes and novel Pt compounds and formulations are under investigation. Liposomal delivery of drugs can reduce patient toxicities while maintaining high treatment doses. Polynuclear Pt agents bind DNA non-covalently in a phosphate clamp-binding motif. Phenanthriplatin initially intercalates with the DNA, improving the efficiency of covalent binding.

Platinum Drug Resistance

Platinum (Pt) drug resistance can be intrinsic (present before treatment) or acquired (upon treatment). Drug response often decreases with subsequent treatments, but retreatment response after a drug holiday is sometimes observed. Non-stable epigenetic mechanisms, such as chromatin-mediated reversible drug-tolerant states, may explain this. Quiescent drug-tolerant cells (DTCs) can restart proliferation after drug exposure.

Chromatin-Repressing Mechanisms in Pt Drug Resistance

Several chromatin-repressing mechanisms have been identified in cells exhibiting platinum drug resistance:

• Demethylation of histone H3 lysine 4 (H3K4) by lysine demethylase 5A (KDM5A).
• Histone tail deacetylation.
• Increase in H3 lysine 27 (H3K27) methylation by enhancer of zeste homologue 2 (EZH2).
• Upregulation of the nucleosome remodeling deacetylase (NuRD) complex, promoting P21 promotor hypermethylation.

Microenvironmental and Immunomodulatory Effects on Platinum Drug Efficacy

The efficacy of platinum (Pt) drug treatment is greatly affected by the tumor microenvironment (TME). The TME has both protective and impairing effects on tumor cells during Pt drug treatment. Cancer-associated fibroblasts (CAFs) export glutathione and cysteines, which can bind and inactivate Pt-based agents, decreasing Pt–DNA adduct formation in tumor cells. Effector T cell production of interferon-γ (IFNγ) abolishes the protective effect of CAFs through STAT1 signaling, which represses the cysteine/glutamate antiporter light chain xCT, reducing glutathione and cysteine export. Increased γ-glutamyl transpeptidase 5 (GGT5) increases extracellular degradation of glutathione. Pt drug-induced interleukin-6 (IL-6) and prostaglandin E2 (PGE2) expression in tumor cells promotes monocyte differentiation towards tumor-promoting M2 macrophages. Oxaliplatin activity relies on myeloid cells stimulated by an intact commensal microbiota, which generate reactive oxygen species (ROS) via NADPH oxidase 2 (NOX2) at the tumor site upon oxaliplatin treatment.

Pt compounds also show immunomodulatory effects on the TME. Pt treatment increases the release of tumor neoantigens (such as calreticulin), high mobility group box 1 (HMGB1) protein, and ATP, which leads to the expansion of tumor-reactive T cells via IL-18 secretion from stimulated antigen-presenting cells (APCs). Cisplatin-induced overexpression of programmed cell death 1 (PD1) ligand 1 (PDL1) on tumor cells and myeloid-derived suppressor cells (MDSCs) inhibits T cell activation, suppressing immune activity in the TME. APCs and tumor cells exposed to Pt agents downregulate the expression of PD1 ligand 2 (PDL2), leading to increased T cell activation. The decrease of PDL2 expression occurs following reduced phosphorylation of STAT6 mediated by Pt drugs. Upregulation of the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway in APCs initiates type I interferon pathways, which can have both tumor-promoting and tumor-suppressing roles.

Cancer Drugs and Biotechnology

Cancer drugs and biotechnology interact, where biotechnology contributes to:

• Identification of therapy targets.
• Development of cancer model systems.
• Development of test system methodologies.
• Development of read-out systems.
• Development of toxicity and efficacy testing models and methods.

Drug Transporters and Chemotherapy

It has to be investigated whether or not inhibition of drug transporters may help in chemotherapy.

Drug Transporters – Two Main Families

Drug transporters consist of two main families:

• ABC (ATP binding cassette) transporter superfamily
  • Based on ATP hydrolysis.
  • Found in the intestinal mucosa, kidney, and blood-brain barrier.
  • Involved in detoxification via efflux.
• SLC (solute carrier) transporter
  • Facilitated ion-connected secondary active transport.
  • Includes organic anion transport (OATP) and organic cation transport (OCTP).
  • Found in the liver and kidney.
    There are approximately 2000 genes coding for transport proteins or transport protein-connected proteins in the human genome.

MDR and Chemotherapy

Multidrug resistance (MDR) involves detoxification and increased compound efflux from the intracellular space to the extracellular space, resulting in drug resistance. This process involves increased metabolite transporter levels, MDR transporter activity, and increased transporter gene expression.

Drug Transport and its Clinical Significance

Drug transport has significant clinical implications.

Multidrug Resistance (MDR) and Drug Sensitivity

If drug resistance decreases, drug sensitivity increases. If either the expression or activity of drug transporters decreases, less drug is needed to effectively kill a tumor cell.

MDR - Multi Drug Resistance and Combined Treatment

Anti-cancer agents combined treatment with a dual drug delivery system improves synergistic anti-cancer activity, reduces MDR and cytotoxicity, and enhances cancer cell targeting while remaining non-cytotoxic.

Active Transport

Active transport requires ATP to open up the efflux transport channels in the cell membrane.

MDR (multidrug resistance) and ABC Transporters

MDR ABC transporters include:

• ABCB1 (MDR1/PgP).
• ABCC10 (MRP7).
• ABCC1 (MRP1).
• ABCG2 (BCRP).
  Several members of the ABC family are involved in drug transport, with specific transporters exporting the most widely used drugs.

Consequences of Drug Transporter Inhibition

Consequences of drug transporter inhibition include:

• Inhibition of all efflux transport in all cells.
• Accumulation of xenobiotics.
• Cell death, potentially leading to patient death.

Main Questions and Answers

1. How would you investigate the mechanism of a drug that you expect to die after treatment?
2. How would you use biotechnology to identify specific proteins?
3. How would you model a specific cancer?
4. How would you investigate drug transport?