Treatment effects of Oxyberberine in SH-SY5Y spheroidal cells against

Oxygen Glucose Deprivation

Presented By
Under Guidance
Neha Krishna Pille
Dr. Manoj P. Dandekar
RT/2022/608
Contents

1. Introduction
2. Literature Review
3. Hypothesis
4. Rationale
5. Aim
6. Objectives
7. Materials and Methods
8. Study Design
9. Expected Outcomes
10. Reference
 Introduction

Neurodevelopmental dysfunction
Reduction of oxygen in tissues
directly associated with brain injury

Hypoxia

Oxygen Glucose Deprivation Activation of HIF-1α, MMP’s,

simulate an ischemic damage angiogenic & growth factors

1. Niu, G., Zhu, D., Zhang, X., Wang, J., Zhao, Y., & Wang, X. (2018). Role of hypoxia-inducible factors 1α (HIF1α) in SH-SY5Y cell autophagy induced by oxygen-glucose deprivation. Medical Science Monitor, 24, 2758–2766.
https://doi.org/10.12659/MSM.905140.
 Activation of Hypoxic Inducible Factor-1α

2. Ziello, J. E., Jovin, I. S., & Huang, Y. (2007). Hypoxia-Inducible Factor (HIF)-1 regulatory pathway and its potential for therapeutic intervention in malignancy and ischemia. Yale Journal of Biology and Medicine, 80(2), 51–60.
 Oxyberberine inhibits HIF-1α expression

Anti-inflammatory
activity in vitro & in
Isoquinoline alkaloid vivo

Inhibits transcription
Berberine Oxyberberine of HIF-1α, MMP-9,
TNF-α, IL-6

Anti-cancer effect Reach at necrotic core

& inhibits angiogenic
factors

3. Pan, Y., Shao, D., Zhao, Y., Zhang, F., Zheng, X., Tan, Y., He, K., Li, J., & Chen, L. (2017). Berberine reverses hypoxia-induced chemoresistance in breast cancer through the inhibition of AMPK-HIF-1α. International Journal of Biological Sciences, 13(6),
794–803. https://doi.org/10.7150/ijbs.18969.
 Introduction to Spheroids

Approximately 2mm3 is characterized by

Aggregated 3D structure can mimic the gradients of metabolites, catabolites &
microenvironment of cell interactions which oxygenation.
are in native tissue or in vivo.

After reaching the size of greater than

It mimic the tumour-behaviour more 2mm3, diffusion supplies insufficient
effectively than regular two dimensional oxygen for tumour so it forms hypoxic
(2D)cell cultures. state.

Macrostructure which dictates a Hypoxia triggers upregulation of the

microenvironment of normal tissue in terms Spheroid hypoxia inducible factor (HIF), which
of oxygenation, perfusion, pH & metabolic initiates the release of proangiogenic
states. factors that stimulates angiogenesis.

4.Jia, M. (2017). HHS Public Access. Physiology & Behavior, 176(3), 139–148. https://doi.org/10.1002/adhm.202001284.Engineered.
 Spheroids mimics BBB

250μm spheroids develop 3 zones :

1. Outer or proliferating zone - It forms cell to cell & cell to
HIF-α is responsible for
75µm in thickness ( Rapidly ECM interaction without any It enables the study of rapid
permeability of BBB &
dividing cells) supportive material, so it screening of brain –
responsible for secreting
2. Intermediate zone – Slower forms tight junctions between penetrating drugs.
pro-angiogenic factors.
division them.
3. Necrotic zone

5.Eilenberger, C., Rothbauer, M., Selinger, F., Gerhartl, A., Jordan, C., Harasek, M., Schädl, B., Grillari, J., Weghuber, J., Neuhaus, W., Küpcü, S., & Ertl, P. (2021). A Microfluidic Multisize Spheroid Array for Multiparametric Screening of Anticancer Drugs
and Blood–Brain Barrier Transport Properties. Advanced Science, 8(11), 1–20. https://doi.org/10.1002/advs.202004856.
 Type of Spheroids

Non-Embedded Spheroids Embedded Spheroids

1. Embedded in Agarose gel – Strain maps of agarose

1. Individual - Without any supportive material
gel on growth of spheroids.

2. Embedded in Matrigel – Reconstituted basement

2. Hanging drop method – Inverting droplet containing
membrane, it forms ductal structure which mimic the
cells, preventing cells from adhering to culture plate
aggregated structure of cells found in vivo.

3. Embedded in collagen – Polymer

3. Liquid Overlay Technique – Non adherent substrate
Polyvinylpyrrolidone, component of ECM, polymer
like agarose/agar or Poly-Hema coated well induces
immobilize the cells at injection site reducing time for
aggregation
spheroid formation.

4. Use of PDMS mold – Offers the control of spheroid

4. Use of Microfluidics – To form complex 3D structure
size & complex pattern

6.Tevis, K. M., Colson, Y. L., & Grinstaff, M. W. (2017). Embedded Spheroids as Models of the Cancer Microenvironment. Advanced Biosystems, 1(10). https://doi.org/10.1002/adbi.201700083.
Literature Review

Table No. 1 - Literature review of OGD induced in different cell line.

Reference Cell line OGD time Outcome

TNF-α, IL-1β, iNOS cause

Li Zhang (2012) BV-2 48 h
apoptosis

OGD – 10 h
Normal condition – 12h Cleaved caspase-3 concentration
Na Lu (2018) SH-SY5Y increased after 10 h of OGD and
HPC – 9 h after 9 h of HPC decreased
Normal condition – 12h

Changed Tight Junction (TJ)

Erica Tornabene (2019) Endothelial cells, Astrocyte 4 h, 24 h, 48 h
proteins localization

ROS production get increased

Young-Jun Li (2021) SH-SY5Y 4 h, 8 h, 12 h, 24 h, 48 h
within 4h of OGD cause cell death
Literature Review

Table No. 2 - Literature review of Berberine treated SH-SY5Y cell in case of OGD.

Reference Key Findings

- Berberine pre-treatment reduced HIF-1α mRNA upregulation,

Wang et al., Brain Res 2018 apoptosis, and cell death following OGD in SH-SY5Y cells

- Berberine protected SH-SY5Y cells against OGD injury by inhibiting

Zhu et al., Metab Brain Dis 2019 HIF-1α accumulation and signaling

- Berberine suppressed OGD-induced HIF-1α expression in SH-SY5Y

Tang et al., Neurochem Res 2021 cells via inhibition of PI3K/Akt/mTOR pathway

- Berberine treatment alleviated OGD-induced cell injury with effects

Song et al., Front Pharmacol 2018 mediated partly through inhibition of HIF-1α and VEGF expression

- Berberine attenuated OGD-triggered inflammatory response and cell

Guo et al., Life Sciences 2018 damage in SH-SY5Y; reduced HIF-1α and downstream signaling
Hypothesis
Rationale

Utilizing SH-SY5Y cell line because it is more sensitive to eliciting HIF-1α under OGD conditions.

HIF-1α rapidly builds up in the nucleus and transactivates hundreds of genes, including receptors, angiogenic and growth
factors, and matrix metalloproteinases.

Certain tumour cells, connective tissue cells, and macrophages release MMP-9 in zymogen.

There is a strong link between MMP-9 activity and brain damage, cerebral edema, cerebral vascular permeability, and blood-
brain barrier permeability.

OBB, a potent anti-inflammatory agent, is the active metabolite of berberine, an isoquinoline alkaloid used as an anticancer
drug.
HIF – α in SH-SY5Y cell line induced by OGD
 Correlation of cerebral ischemia with OGD in cell line

Cerebral
Ischemia

Brain damage

HIF – α

TIMP Destroy basement

membrane

Pro-MMP9

MMP9
BBB permeability
Aim
To target HIF-1α in order to investigate the impact of Oxyberberine on Oxygen Glucose Deprivation induced neuroblastoma
cell spheroids.

Objectives

1. To prepare Polydimethylsiloxane (PDMS) and Curing agent devices for formation of SH-SY5Y cell spheroids.

2. To develop the hypoxia in SH-SY5Y cell spheroids with the help of OGD chamber.

3. To determine the effect of OBB on HIF-1α and MMP-9 production in hypoxic SH-SY5Y cell spheroids.
 Materials and Methods

SH-SY5Y Cell

1.Neuroblast Cell

2. Obtained from metastatic bone tumour

from 4yrs female child.

3. Characteristics –
I. Adherant

II. Neuroblast like

Morphology of SH-SY5Y
phenotype is sensitive for HIF- 1α .

III. It has neuronal markers

7.Feles, S., Overath, C., Reichardt, S., Diegeler, S., Schmitz, C., Kronenberg, J., Baumstark-Khan, C., Hemmersbach, R., Hellweg, C. E., & Liemersdorf, C. (2022). Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for
Functional Studies. Methods and Protocols, 5(4). https://doi.org/10.3390/mps5040058.
 Formation of Spheroids using Polydimethylsiloxane

1. Polydimethylsiloxane is colourless silicon based polymer.

2. Curing agent – Used for fabricating PDMS

3. Mixture of PDMS & Curing agent is responsible for formation of moulds by

adding in Aggrewell 400µ.

4. AggreWell containing 400µ grooves which enables growth of cells in

aggregated 3D structure.
PDMS + Curing agent AggreWell 400µ
(10:1)

5. By inverting that moulds in mixture of PDMS & Curing agent which is

responsible for formation of spheroids.

8.Khot, M. I., Levenstein, M. A., de Boer, G. N., Armstrong, G., Maisey, T., Svavarsdottir, H. S., Andrew, H., Perry, S. L., Kapur, N., & Jayne, D. G. (2020). Characterising a PDMS based 3D cell culturing microfluidic platform for screening chemotherapeutic
drug cytotoxic activity. Scientific Reports, 10(1), 1–13. https://doi.org/10.1038/s41598-020-72952-1.
 Oxygen Glucose Deprivation

1. Commonly used for inducing ischemic injury in vitro.

2. Treatment consist of replacement of standard medium with a

hypoxic N2/CO2 equilibrated culture medium without glucose.

3. Incubation of cells in a hypoxic chamber having the same N 2/CO2

gas combination.

4. The replacement of the culture medium is performed by

aspirating up the medium & subsequently adding the new medium
to culture system.

5. Components – OGD Chamber

I. Modular Incubator Chambers
II. Gas Flow Meters
III. Flow – Through Oxygen Monitor
IV. Data – logging Oxygen & Carbon Dioxide Monitor

9.Tornabene, E., Helms, H. C. C., Pedersen, S. F., & Brodin, B. (2019). Effects of oxygen-glucose deprivation (OGD) on barrier properties and mRNA transcript levels of selected marker proteins in brain endothelial cells/astrocyte co-cultures. PLoS ONE, 14(8),
1–20. https://doi.org/10.1371/journal.pone.0221103.
Study Design
Work Done

Formation and analysis of spheroid formation using light microscopy

a. Mold formation and devices making.

PDMS Molds PDMS Devices

b. Impregnation of shy5y cells into devices which further

continues to spheroid formation.

SH-SY5Y cell spheroids in SH-SY5Y cell spheroid

device
Future Plan

1. Development of SH-SY5Y cell embedded and non-embedded spheroids is to be done.

2. Spheroids are to be exposed with OGD chamber and further treated with OBB.

3. After the development of hypoxia in spheroids, cell viability will be assessed using the MTT assay.

4. Molecular parameters i.e. HIF-1α, MMP-9 are to be accessed with the aid of Western blot, flow cytometry, RTPCR, and cell
invasion assay.
Expected Outcomes
1. A 3D structure called a spheroid can imitate the in vivo or native tissue microenvironment by creating three distinct zones
which resemble the BBB with formation of ECM.

2. Due to OGD HIF-1α may activate MMP-9 in SH-SY5Y cell spheroids, leads to brain damage from hypoxia & ischemia.

3. Under hypoxic condition, MMP-9 breaks ECM, allowing cells invade from spheroid structure & cause death.

4. Oxyberberine is potent anti-inflammatory agent which reduce HIF-1α production.

Reference
1. Niu, G., Zhu, D., Zhang, X., Wang, J., Zhao, Y., & Wang, X. (2018). Role of hypoxia-inducible factors 1α (HIF1α) in SH-SY5Y cell autophagy induced by oxygen-glucose deprivation.
Medical Science Monitor, 24, 2758–2766. https://doi.org/10.12659/MSM.905140.
2. Ziello, J. E., Jovin, I. S., & Huang, Y. (2007). Hypoxia-Inducible Factor (HIF)-1 regulatory pathway and its potential for therapeutic intervention in malignancy and ischemia. Yale
Journal of Biology and Medicine, 80(2), 51–60.
3. Pan, Y., Shao, D., Zhao, Y., Zhang, F., Zheng, X., Tan, Y., He, K., Li, J., & Chen, L. (2017). Berberine reverses hypoxia-induced chemoresistance in breast cancer through the inhibition
of AMPK-HIF-1α. International Journal of Biological Sciences, 13(6), 794–803. https://doi.org/10.7150/ijbs.18969.
4. Jia, M. (2017). HHS Public Access. Physiology & Behavior, 176(3), 139–148. https://doi.org/10.1002/adhm.202001284.Engineered.
5. Eilenberger, C., Rothbauer, M., Selinger, F., Gerhartl, A., Jordan, C., Harasek, M., Schädl, B., Grillari, J., Weghuber, J., Neuhaus, W., Küpcü, S., & Ertl, P. (2021). A Microfluidic
Multisize Spheroid Array for Multiparametric Screening of Anticancer Drugs and Blood–Brain Barrier Transport Properties. Advanced Science, 8(11), 1–20.
https://doi.org/10.1002/advs.202004856.
6. Tevis, K. M., Colson, Y. L., & Grinstaff, M. W. (2017). Embedded Spheroids as Models of the Cancer Microenvironment. Advanced Biosystems, 1(10).
https://doi.org/10.1002/adbi.201700083.
7. Feles, S., Overath, C., Reichardt, S., Diegeler, S., Schmitz, C., Kronenberg, J., Baumstark-Khan, C., Hemmersbach, R., Hellweg, C. E., & Liemersdorf, C. (2022). Streamlining Culture
Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies. Methods and Protocols, 5(4). https://doi.org/10.3390/mps5040058.
8. Khot, M. I., Levenstein, M. A., de Boer, G. N., Armstrong, G., Maisey, T., Svavarsdottir, H. S., Andrew, H., Perry, S. L., Kapur, N., & Jayne, D. G. (2020). Characterising a PDMS
based 3D cell culturing microfluidic platform for screening chemotherapeutic drug cytotoxic activity. Scientific Reports, 10(1), 1–13. https://doi.org/10.1038/s41598-020-72952-1.
9. Tornabene, E., Helms, H. C. C., Pedersen, S. F., & Brodin, B. (2019). Effects of oxygen-glucose deprivation (OGD) on barrier properties and mRNA transcript levels of selected marker
proteins in brain endothelial cells/astrocyte co-cultures. PLoS ONE, 14(8), 1–20. https://doi.org/10.1371/journal.pone.0221103.
10. Montalto, A. S., Currò, M., Russo, T., Ferlazzo, N., Caccamo, D., Ientile, R., Romeo, C., & Impellizzeri, P. (2020). CO 2Pneumoperitoneum Effects on Molecular Markers of Tumor
Invasiveness in SH-SY5Y Neuroblastoma Cells. European Journal of Pediatric Surgery, 30(6), 524–528. https://doi.org/10.1055/s-0039-1700547.
11. Zhang, L., Dong, L. Y., Li, Y. J., Hong, Z., & Wei, W. S. (2012). The microRNA miR-181c controls microglia-mediated neuronal apoptosis by suppressing tumor necrosis factor. Journal
of Neuroinflammation, 9(1), 1. https://doi.org/10.1186/1742-2094-9-211.
12. Choi, J. Y., Jang, Y. S., Min, S. Y., & Song, J. Y. (2011). Overexpression of MMP-9 and hif-1α in breast cancer cells under hypoxic conditions. Journal of Breast Cancer, 14(2), 88–95.
https://doi.org/10.4048/jbc.2011.14.2.88.
13. Lin, S., Tsai, S. C., Lee, C. C., Wang, B. W., Liou, J. Y., & Shyu, K. G. (2004). Berberine inhibits HIF-1α expression via enhanced proteolysis. Molecular Pharmacology, 66(3), 612–
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